Phosphatase inhibitor

Tissue samples were lysed in RIPA lysis buffer containing a protease inhibitor and a phosphatase inhibitor (Applygen Technologies Inc., Beijing, China). The protein concentrations of all samples were measured using the BCA Protein Assay Kit (Applygen Technologies Inc.). Protein samples (equal amounts per lane) were run on Criterion SDS–PAGE gels (Bio–Rad) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Darmstadt, Germany). The transfer membranes were blocked in blocking buffer (TBS with 0.1% Tween 20 (TBST) and 5% skim milk) for 1 h at RT followed by incubation with the following primary antibodies: anti-NG2 (1 : 1000, ab129051, Abcam), anti-Olig2 (1 : 500, MABN50, Millipore), anti-GST-pi (1 : 800, 312, MBL), anti-MBP (1 : 1000, GB12226, Servicebio), anti-GEF (1 : 1000, ab201687, Abcam), anti-Rac1 (1 : 500, ARC03, Cytoskelet), anti-phospho-Rac1 (1 : 1000, 2641, CST), anti-Cdc42 (1 : 1000, 2466S, CST), anti-MRCKα (1 : 1000, 81681S, CST), anti-PAK1 (1 : 1000, 2602, CST), anti-Tubulin (1 : 20,000, 66031-1, Proteintech), anti-GAPDH (1 : 20,000, 60004-1, Proteintech), and the corresponding HRP-conjugated anti-IgG antibodies. Finally, the protein bands were detected using a gel chemiluminescence imaging system and quantified by determining the grayscale values using ImageJ. All experiments were repeated three to four times.

Cell lysates were prepared in RIPA lysis buffer containing phosphatase inhibitor (Applygen Technologies, Beijing, China) as previously described (Zhang et al., 2016 (link)). Equal amounts of protein were separating using SDS-PAGE, then transferred to PVDF membranes (Millipore, MA, USA). After blocking, the membranes were incubated with the following primary antibodies at 4 °C overnight: Phospho-NF-κB p65 (Ser536), Phospho-Stat3 (Tyr705), Phospho-IKKα/β (Ser176/180), Phospho-IκBα (Ser32), IKKβ, Stat3, NF-κB p65, IκBα, PD-L1, and GAPDH (Cell Signaling Technology, MA, USA). Proteins were detected using HRP-conjugated secondary antibodies and Immobilon™ Western Chemiluminescent HRP substrate (Millipore, MA, USA). Protein band density was quantified using a ChemiDoc ™ Imaging System (Bio-Rad, CA, USA).

Renal tissues or cells were homogenized in RIPA lysis buffer (Mei5bio) containing 4% protease inhibitor cocktail (Roche, Basel, Switzerland) and 1% phosphatase inhibitor (Applygen, Beijing, China). Total protein concentration was detected by BCA kit (Pierce, Rockford, IL, USA) after ultrasonic cracking. Identical amounts of protein samples were electrophoresed on polyacrylamide gels and electrotransferred to polyvinylidene difluoride membranes, and then the membranes were incubated with antibodies against b-actin (Santa Cruz), COX-2, IL-6, TLR4 (ABclonal, Wuhan, China), iNOS, MyD88 (Abcam, Cambridge, UK), caspase-3, cleaved caspase-3, caspase-8, cleaved caspase-8, NF-kB, p-NF-kB (Immunoway, Plano, TX, USA), at 4 °C overnight. Then, goat anti-rabbit IgG or goat anti-mouse IgG (EASYBIO, Beijing, China) were added and the blots were developed with ECL plus kit (Biodragon, Beijing, China) and were visualized with a chemiluminescence detection system (Syngene, GeneGnome XRQ, Cambridge, UK). Quantitation was performed by scanning and analyzing the intensity of the hybridization bands and the data were analyzed with Image J software.

The protein lysis buffer containing a phosphatase inhibitor (Applygen Technologies Inc., Beijing, China) was used to harvested cells. The cell suspensions were centrifuged at 4°C for 30 min with a speed of 12,000 × g. The BCA Protein Assay (CWBIO, Beijing, China) was used to determine the protein concentration, and each lane was loaded with equal aliquots of the total protein (20 μg). The sample lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA), blocked in blocking sodium (Beyotime, Shanghai, China) for 1 h, and probed with the following antibodies at 4°C overnight: DSPP (1 : 1000; Santa Cruz Technology, Santa Cruz, CA), DMP1 (1 : 1000; Bioss, Beijing, China), p38, p-p38, and β-actin (1 : 10000; Cell Signaling Technology, Beverly, MA, USA). The membrane was incubated at room temperature for 1 h with horseradish peroxidase- (HRP-) conjugated antirabbit immunoglobulin. The protein expression was detected using a Western enhanced chemiluminescence blotting kit (ECL, SOLIBRO, Beijing, China).

The cells were harvested with protein lysis buffer containing a phosphatase inhibitor (Applygen Technologies Inc., Beijing, China). The cell suspensions were centrifuged at 4 °C for 30 min at 12,000 × g. The protein concentration was determined using bicinchoninic acid (BCA) Protein Assay (CWbio, Beijing, China) and each lane was loaded with equal aliquots of total protein (20 μg). The lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA), blocked in 5% bovine serum albumin for 2 h, and probed with antibodies against the following at 4 °C overnight: IGFBP7 (sc-365293, 1:1000, Santa Cruz Biotechnology, CA, USA), DSPP (sc-73632, 1:1000, Santa Cruz Biotechnology, CA, USA), and β-actin (#4970, 1:10,000, Cell Signaling Technology, Beverly, MA, USA). The membrane was incubated at room temperature for 1 h with horseradish peroxidase-conjugated anti-rabbit immunoglobulin. Protein expression was detected using a western enhanced chemiluminescence blotting kit (Solibro, Beijing, China).

Total proteins were extracted with RIPA-Buffer (Beyotime Biotechnology, Shanghai, China) supplemented with 10 mM PMSF (Sigma-Aldrich, St Louis, MO, USA) and phosphatase inhibitor (Applygen, Beijing, China). BCA method was carried out to quantify protein concentrations. 40 μg proteins were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to polyvinylidene fluoride (PVDF) membranes. Then, the membrane was blocked with 5% nonfat milk in Tris-buffered saline and 0.1% Tween 20 for 1 h. Subsequently, the membranes were incubated with primary antibodies at 4°C overnight and washed with TBST buffer for 5 times. The membranes were incubated with Horseradish Peroxidase- (HRP-) conjugated secondary antibodies for 2 h. An enhanced chemoluminescence (ECL) system kit (Beyotime, Shanghai, China) was used to visualized using an automatic chemoluminescence image analysis system (Tanon, Shanghai, China). Optical densities (OD) value was analyzed by ImageJ software (NIH, Bethesda, MD, USA).