1. Reagents and solutions RIPA buffer: 20mM Tris (pH 7.5), 1mM Na2EDTA, 1mM EGTA, 2.5mM sodium pyrophosphate, 150mM NaCl, 1mM beta-glycerophosphate disodium salt hydrate, 1mM Na3VO4, 1µg/ml leupeptin hemisulfate salt, 1% sodium deoxycholate, 1% Triton X-100, 1mM PMSF

Experimental procedure — Preparation of cell lysis solution

1) Cell collection: Choose logarithmic phase cells, wash cells with pre-cooled PBS, centrifuge at 3000rpm for 5min, discard supernatant;

2) Cell lysis: Gently tap the cell pellet to disperse, then add an appropriate amount of pre-cooled RIPA buffer and PMSF for lysis (for a 100mm diameter cell culture dish, add 100–200μl lysis buffer, for 1ml RIPA buffer add 10μl PMSF), place on ice, gently blow the cell suspension apart, leave in a 4°C refrigerator or ice-water mixture for lysis for 40–60min;

3) Sonication: First observe the lysis condition, if the liquid is clear, indicating sufficient lysis and no need for sonication; otherwise, if lysis is insufficient, proceed to sonication. The method is as follows: Set parameters: 10–30 cycles, each lasting 5–10 seconds, take the cell lysis suspension out from the 4°C refrigerator or ice bath and place it in an ultrasonicator (sonication on ice). After sonication, observe whether the cell lysis liquid is clear; if turbid, sonicate again;

4) Centrifugation and protein concentration measurement: Centrifuge at 13000rpm, 4°C for 8min, collect the supernatant. Take another 10μl sample and measure the protein concentration using the Coomassie Brilliant Blue method;

5) Cell lysis solution treatment: Add an appropriate amount of 5*Loading Buffer, mix well, then boil for 5–10min, take out 0.5ml sample for electrophoresis and WB detection, store the rest at -20°C.

6) Experimental procedure — Preparation of tissue lysis solution

7) Tissue collection: Take fresh tissue or tissue from -80°C freezer/liquid nitrogen required for lysis solution preparation, after tissue thaws place it in a culture dish with PBS, use tweezers to clean residual blood in the tissue and remove as much fat and outer membrane as possible;

8) Place the cleaned tissue in a culture dish, cut the tissue into small pieces with scissors (as small as possible);

9) Lysis: Transfer the chopped tissue pieces to a mortar, after grinding a certain number of times in an ice bath, add an appropriate amount of pre-cooled RIPA buffer and PMSF and continue grinding until no obvious chunks of tissue remain (generally 1ml RIPA buffer with 10μl PMSF);

10) Transfer the ground tissue lysis suspension to a clean centrifuge tube, rinse the mortar with a small amount of RIPA buffer and PMSF and pour into the centrifuge tube, place in an ice bath for 40–60min, fully lysing, shake the centrifuge tube or tap it several times intermittently;

11) Sonication: First observe the lysis condition, if the liquid is clear, indicating sufficient lysis and no need for sonication; otherwise, if lysis is insufficient, proceed to sonication. The method is as follows: Set parameters: 10–30 cycles, each lasting 5–10 seconds, take the cell lysis suspension out from the 4°C refrigerator or ice bath and place it in an ultrasonicator (sonication on ice). After sonication, observe whether the cell lysis liquid is clear; if turbid, sonicate again;

12) Centrifugation and protein concentration measurement: Centrifuge at 13000rpm, 4°C for 8min, collect the supernatant (carefully avoid the fat on the surface and tissue debris at the bottom of the tube), take another 10μl sample and measure the protein concentration using the Coomassie Brilliant Blue method; Tissue lysis solution treatment: Add an appropriate amount of 5*Loading Buffer, mix well, then boil for 5–10min, take out 0.5ml sample for electrophoresis and WB detection, store the rest at -20°C.