The inserted DNA for all plasmids except pGL4.35 were synthesized and cloned into respective vectors by Sangon corporation.

(1) Retrieve competent DH5α Escherichia coli cells from the -80°C freezer and thaw them on ice (do not vortex or pipette vigorously).

(2) Remove the plasmids to be amplified from the -20°C freezer and place them on ice for thawing.

(3) Label microcentrifuge tubes with the respective plasmid names.

(4) Add 30 μL of DH5α Escherichia coli to each labeled microcentrifuge tube.

(5) Add 1 μL of each thawed plasmid to the corresponding tube, then return the plasmids to the -20°C freezer immediately after use.

(6) Incubate the tubes containing the mixture on ice for 30 minutes.

(7) Transfer the tubes to a preheated 42°C heat block for a 90-second heat shock, then immediately place them back on ice for incubation for at least 2 minutes.

(8) Remove ampicillin from the -20°C freezer and thaw by warming in hand.

(9) Retrieve LB (-) medium (sterile) from the 4°C refrigerator. Prepare a test tube rack, alcohol lamp, and culture tubes (pre-labeled with the corresponding plasmid names). Ignite the alcohol lamp, and under aseptic conditions near the flame, add 5 mL of LB (-) medium and 5 μL of ampicillin to each culture tube.

(10) Remove the ice-incubated plasmid mixtures and transfer the entire volume into the corresponding labeled culture tubes (do not fully tighten the caps).

(11) Place the 6 culture tubes on a shaking incubator and incubate at 37°C with vigorous shaking (200–250 rpm) for 12–16 hours.

(1) Label 15 mL centrifuge tubes with the names of the plasmids to be extracted.

(2) Retrieve the culture tubes from the shaker and transfer the bacterial cultures to the corresponding 15 mL centrifuge tubes. Centrifuge at 13,000 rcf for 1 minute (ensure symmetrical placement in the centrifuge, same for subsequent steps).

(3) Remove the centrifuged tubes, carefully discard the supernatant, and resuspend the bacterial pellet by gentle pipetting with the residual medium.

(4) Label microcentrifuge tubes with the names of the plasmids to be extracted, and transfer the resuspended bacterial solution to the corresponding tubes.

(5) Centrifuge the microcentrifuge tubes at 13,000 rcf for 1 minute. After centrifugation, remove the tubes and discard the supernatant.

(6) Add 250 μL of Buffer P1 (supplemented with RNase A, retrieved from 4°C storage) to each tube, then resuspend the bacterial pellet by vortexing at maximum speed until no clumps remain.

(7) Add 250 μL of Buffer P2 to the resuspended solution and invert the tube 8–10 times until the solution becomes clear.

(8) Add 350 μL of Buffer P3 to each tube and immediately invert 8–10 times.

(9) Centrifuge the tubes at 13,000 rcf for 10 minutes.

(10) Place a HiPure DNA Mini Column Ⅳ (hereinafter referred to as "column") into a collection tube. Carefully transfer the supernatant to the column and centrifuge at 13,000 rcf for 60 seconds.

(11) Discard the filtrate, return the column to the collection tube, add 500 μL of Buffer PW1 to the column, and centrifuge at 13,000 rcf for 60 seconds.

(12) Discard the filtrate, return the column to the collection tube, add 600 μL of Buffer PW2 (pre-diluted with absolute ethanol) to the column, and centrifuge at 13,000 rcf for 60 seconds.

(13) Discard the filtrate, return the column to the collection tube, add 300 μL of Buffer PW2 (pre-diluted with absolute ethanol) to the column, and centrifuge at 13,000 rcf for 60 seconds.

(14) Discard the filtrate, return the column to the collection tube, and centrifuge at 13,000 rcf for 60 seconds to dry the column.

(15) Place the column into a sterile 1.5 mL microcentrifuge tube. Add 50 μL of Elution Buffer or sterile water to the center of the column membrane. Allow to stand for 1 minute, then centrifuge at 13,000 rcf for 60 seconds to elute the plasmid DNA.

(16) Determine the concentration and purity of the extracted plasmid DNA using a microvolume spectrophotometer: load 1 μL of the eluted sample, and the instrument-connected software will generate quantitative data for nucleic acid concentration (ng/μL) and A260/A280 ratio.

（1）Take out the 293T cells cultured in the 24-well plate from the incubator, aspirate the medium, and wash once with 100 µL of 1×PBS.

（2）Add 100 µL of 1×PBS to each well.

（3）Add 100 μL of 1×Reporter Lysis Buffer (diluted with 5×Reporter Lysis Buffer and ddH₂O).

（4）Pipette gently multiple times to transfer to a 1.5 mL EP tube, and mark it well.

（5）Vortex and let stand for 5 minutes.

（6）Measure the luciferase value

① Set up the FB12 Sirius software, select Dual Assay for Protocol types, select Dual-Luciferase Assay for Runnable protocols, and click Run to start the experiment.

② Pipette repeatedly to mix well, then put it into the Sivius single-tube chemiluminescence detector for detection. The detector will automatically detect the intensity of fluorescence, which will be displayed numerically on the FB12 Sirius software, and the first data will be obtained from the measurement.

③ Add 10 µL of renilla and 1 µL of terminator mixture to each tube (prepared freshly before use, and the stock solution is stored at -80°C). Pipette to mix well, then put it into the Sivius single-tube chemiluminescence detector for detection, and the second data will be obtained from the measurement.

④ Copy all data, export it to an Excel spreadsheet, and perform normalization processing and analysis on the data using Stop&Glo data.

(1)Rinse the glass plates thoroughly, followed by a final rinse with ddH₂O, and allow them to air-dry at room temperature. Once dry, carefully align a set of glass plates tightly and fix them on a dedicated gel casting stand.

(2)Prepare the separating gel by sequentially adding the reagents listed in the table below into a 50 ml centrifuge tube:

After repeatedly inverting and mixing gently, slowly add approximately 7 mL of the mixture into the gap between the glass plates (take care to minimize bubble formation). Then carefully add 2 mL of absolute ethanol to seal the gel—at this point, a distinct boundary layer can be observed between the absolute ethanol and the separating gel.

(3)Allow the mixture to stand at room temperature until the separating gel solidifies. Discard the absolute ethanol layered on top of the gel, then invert the glass plate assembly onto filter paper to dry.

(4)Prepare the stacking gel by sequentially adding the reagents listed in the table below into a 50 mL centrifuge tube：

After mixing the solution thoroughly, add 3 mL of it into the gap above the separating gel in the glass plate assembly, then gently insert a cleaned gel comb into the stacking gel.

After allowing the gel to solidify at room temperature, remove the SDS-PAGE gel from the gel casting stand. Carefully pull out the gel comb, rinse off any residual gel on the surface of the glass plates with running water, and use the gel immediately or store it in a sealed bag at 4℃.

(1) Fix the SDS-PAGE gel in the corresponding position of the electrophoresis tank, fill it with freshly prepared electrophoresis buffer, and ensure the liquid level covers the upper edge of the gel.

(2) Add an appropriate volume of protein Marker and protein loading buffer into the gel wells in sequence, turn on the power, cover the electrophoresis tank lid, and perform electrophoresis at a constant voltage of 90V for 30 minutes.

(3) At this point, prepare the transfer buffer in advance (freshly prepared and used immediately), and pre-cool it in a 4°C or -20°C refrigerator.

(4) Subsequently, switch to a constant voltage of 120V and continue electrophoresis for approximately 60 minutes. The electrophoresis time can be adjusted according to the real-time position of the corresponding bands of the protein Marker and the molecular weight of the target protein.

(5) After electrophoresis is completed, open the transfer夹板 and place it in a tray, pour the pre-cooled transfer buffer into an enamel tray, take out the SDS-PAGE gel from the electrophoresis tank, gently pry open the glass plate with a plastic pry plate, and take out the gel block and place it on the filter paper near the black side of the transfer clamp.

(6) Briefly soak the cut PVDF membrane in methanol to activate the positive charge groups on the membrane, then lay it flat over the gel block, and use a plastic pry plate to drive out air bubbles between the membrane and the gel block.

(7) Cover with filter paper and sponge in sequence, use a plastic pry plate to drive out air bubbles again, close the transfer clamp and clamp it tightly to form a "sandwich" structure of "transfer clamp transparent side - sponge - filter paper - PVDF membrane - PAGE gel - filter paper - sponge - transfer clamp black side".

(8) Insert the transfer clamp into the transfer tank in the direction of "black to black" (the black side of the transfer clamp is aligned with the black side of the transfer tank), put in the ice grid, fill it with fresh transfer buffer, place the transfer tank in an ice-water mixture bath, and cover the transfer tank lid.

(9) Turn on the power, with a constant current of 290mA, and transfer for 90 minutes.

(10) After the transfer is completed, open the transfer clamp to take out the PVDF membrane, with the side close to the gel block facing up, mark the upper right corner, put it into the incubation box, pour 5% skimmed milk, and seal it on a shaker at room temperature for 1 hour with slow shaking.

(11) After sealing, recover the skimmed milk, add an appropriate amount of 0.1% TBST membrane washing solution, shake quickly on the shaker for 5 minutes, discard the membrane washing solution, and repeat 3 times to rinse off the residual milk.

(12) Pour in the corresponding primary antibody, place it on a shaker in a 4°C cold storage, and incubate overnight with slow shaking (the antibody incubation time can be adjusted according to experimental experience).

(13) After the primary antibody incubation is completed, recover the primary antibody, first quickly rinse the residual primary antibody in the box with membrane washing solution, then add an appropriate amount of membrane washing solution, and wash the membrane on the shaker with quick shaking for 8 minutes each time, a total of three times.

(14) Add the corresponding secondary antibody incubation solution (prepared with 5% skimmed milk) according to the species source of the primary antibody, and incubate on a shaker at room temperature with slow shaking for 1 hour.

(15) After incubation, discard the secondary antibody, add an appropriate amount of membrane washing solution, shake quickly on the shaker for 8 minutes, and wash the membrane three times.

(16) Pipette equal volumes of developing solution A and B into a 1.5mL EP tube, mix well, drop the developing solution evenly on the membrane to cover the entire membrane, and after incubating for an appropriate time, perform development with a multi-functional imager.

(17) The development results are scanned and analyzed with ImageJ or Photoshop software. After measuring the grayscale value of the target band, first normalize it with the grayscale value of the internal reference, and then compare the changes in the expression level of the target protein.