Proteinase inhibitors cocktail

For endogenous AGO1 and AGO2 IP, one 10 cm dish of each cell line was lysed in 1 ml modRIPA buffer (10 mM Tris-cl pH 7.0, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and 0.1% SDS) supplemented with proteinase inhibitors cocktail (Roche, 11873580001). Cell lysate was incubated with 50 µl SureBeads Protein G Magnetic Beads (Bio-Rad) plus 5 µg mouse anti-AGO1(2A7) (Wako, 015-22411) or anti-AGO2(4G8) (Wako, 015-22031) monoclonal antibody at 4 °C overnight with rotation. After washing 5 times with TBS buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl) at room temperature, the beads were lysed in 1 ml Trizol (Life Technologies,15596-018) for RNA extraction. For ectopic AGO1 and AGO2 IP, cells were lysed 48 h post-transfection and followed the same protocol except using anti-Flag M2 beads (Sigma, M8823) instead.

Cells or fresh tissues were lysed on ice using RIPA lysis buffer (Solarbio, Beijing, China) with freshly added proteinase inhibitors cocktail (Roche). The protein extracts were clarified and quantified by Pierce BCA Protein Assay (Thermo Scientific). Western blot was performed according to our published procedure^{24 (link)}. The primary antibodies used as follows: anti-TBX3 (#42-4800, Invitrogen, 1:50), anti-Tubulin (#T5168, Simga, 1:10000), anti-BRAF (#14814, CST, 1:2000), anti-c-Jun (#9165, CST, 1:2000), anti-ERK1/2 (#4695, CST, 1:2000), anti-p-ERK1/2 (#4370, CST, 1:2000), anti-JunB (#3753, CST, 1:1000), anti-c-Fos (#2250, CST, 1:1000), anti-GAPDH (#5174, CST, 1:1000), anti-β-Actin (#3700, CST, 1:1000), anti-p65 (#8242, CST, 1:1000), anti-p-p65 (#3033, CST, 1:1000), anti-IKKβ (#8943, CST, 1:1000), anti-p-IKKα/β (#2697, CST, 1:1000), TLR2 (#bs-1019R, Bioss, 1:1000), anti-AKT (#4691, CST, 1:2000), anti-p-AKT (#4060, CST, 1:1000), anti-p38 (#8690, CST, 1:1000), anti-p-p38 (#4511, CST, 1:1000), anti-JNK (#9252, CST, 1:1000), anti-p-JNK (#9255, CST, 1:1000), anti-STAT3 (#9139, CST, 1:1000), anti-p-STAT3 (#9145, CST, 1:1000).

Podocytes were washed with cold PBS and then lysed with 150 μl of RIPA buffer containing proteinase inhibitors cocktail (Roche) and phosphatase inhibitors. The lysates were incubated on ice and then centrifuged 12,000 g for 15 min at 4°C. The supernatants were transferred to fresh tubes and then subjected to protein concentration measurement with BCA protein kit (Bio-Rad). After mixed with loading buffer, the samples were boiled at 98°C for 5 min. 10 or 8% SDS-PAGE was used to fractionate the samples, and semi-dry transfer system (Bio-rad) was used to transfer the protein from the gel to PVDF membrane. The blot was blocked with 5% milk in TBST solution (20 mM Tris-HCl, PH 7.14, 150 mM NaCl, 0.1% Tween-20) for 60 min at room temperature, and then incubated with an antibody overnight at 4°C. After washed with TBST for 3 times, the blot was incubated with HRP-labeled secondary antibody for 1 h at room temperature. After washed, ECL system (Millipore) was used to detect the protein. Cell lysates were prepared using radioimmunoprecipitation assay (RIPA) buffer containing a protease inhibitor cocktail (Roche) and a phosphatase inhibitor. The blot was incubated with a primary antibody of interest.

Tissue mitochondria isolation was performed via differential centrifugation. Mitochondria Isolation Buffer (MIB) (210 mM mannitol, 70 mM sucrose, 5 mM HEPES, 1 mM EGTA, 0.5% BSA, pH 7.2) supplemented with proteinase inhibitors cocktail (Roche) was used to homogenize approximately 100 mg of tissue in a 1 ml glass mortar and pestle, manually, on ice. The homogenate was centrifuged (1000 g, 5 min, 4°C), the supernatant was kept on ice and the pellet was resuspended in MIB and centrifuged again (1000 g, 5 min, 4°C). The two supernatants were mixed and centrifuged (12000 g, 10 min, 4°C) twice to acquire the mitochondrial pellet and the cytoplasmic fraction. The mitochondrial pellet was washed with 500 μl MIB and after centrifugation was resuspended in a minimal volume of MIB. Protein concentration was determined using the Bradford method (Bio-Rad Protein Assay).