Ecl kit

Tissues of LVAW were taken from each experimental miniature swine, cut into pieces at 4°C in a mortar, homogenized, and centrifuged at 10,000 rpm at 4°C for 15 min. A BCA kit was used to quantify the total protein concentration of the supernatant. Samples were mixed with distilled water and 5× SDS-loading buffer. Proteins were separated using 10% SDS polyacrylamide gel electrophoresis. Proteins were transferred onto a nitrocellulose membrane. GRP78 and GADD153/CHOP antibodies (both 1:1000) were added and incubated at 4°C overnight. After washing the membrane, sheep anti-rabbit IgG (1:1000) was added at room temperature and incubated for 2 h. Antibodies against β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:2000) were used for normalization. Bands were revealed using an ECL kit (Cell Signaling, Danvers, MA, USA). The image analysis software Image-J was used to analyze the protein bands for integrated optical density (IOD). IOD = average optical density value × area. Target protein IOD value/β-actin IOD ratio reflects the level of target protein expression.

All snap frozen tissue specimens and cells were lysed by RIPA buffer (Thermo Scientific) containing a protease inhibitor cocktail (Roche, Welwyn Garden, Swiss, UK). Samples were centrifuged at 12 000 rpm for 15 min at 4 °C, and protein concentrations were quantified using the bicinchoninic acid method (Sigma-Aldrich). Thirty micrograms of protein per sample were denatured in loading buffer prior to 10% or 15% SDS-PAGE electrophoreses, and then transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). Non-specific binding was blocked by incubating the membranes in 5% non-fat milk for 1 h at room temperature. Membranes were then incubated overnight at 4 °C with either rabbit anti-MDIG (1:200; Abcam, Cambridge, MA, USA, ab155335), rabbit anti-IKZF1 (1:200; Santa Cruz, SC-13039), rabbit anti-H3K9me3 (1:200; Abcam, ab8898), rabbit anti-H3K9me2 (1:200; Abcam, ab115159) and rabbit anti-H3K9me1 (1:500; Abcam, ab194693) primary antibodies. After washing in phosphate-buffered saline-Tween-20 (PBST), the membranes were incubated with horseradish peroxidase-goat anti-rabbit antibody (1:3000; Sigma-Aldrich, A0545) at room temperature for 2 h, then washed again in PBST. Western blots were visualized using enhanced chemiluminescence (ECL) detection reagent with an ECL kit (Cell Signaling Technology, Danvers, MA, USA). Antibodies are shown in Supplementary Table 1.

Western blot analysis was performed as previously described 21 (link), tissues and cells were washed with PBS, and lysed with RIPA lysis buffer including protease lysis inhibitor (Roche USA) and phosphatase inhibitor (Roche USA). A BCA protein assay kit (Thermo Scientific, USA) was used to measure protein concentration. 30ug protein extract was separated with a 10% SDS-polyacrylamide gel and transferred to a PVDF membrane (microwell). The membrane was blocked with TBST buffer containing 5% skim milk at 37 °C for 2 hours, incubated with primary antibody at 4 °C overnight, and incubated with peroxidase-conjugated secondary antibody for 2 hours. The signal was detected using ECL kit (Cell Signaling Technology, 12757) and photographed. Bands were quantified using gray-scale analysis software (Bio-Rad). GAPDH expression is used as a loading control to standardize the expression of other proteins. The main antibodies are as follows: anti-ADNP (1: 1000, Proteintech, USA), anti-GAPDH, N-Cadherin, Vimentin, Snail, E-Cadherin, β- Catenin, Claudin-1, TGF-β, TGF-βR1, Smad2/3, p-Smad2/3 (1: 1000, Cell Signaling Technology, USA). The secondary antibodies are as follows: HRP-goat anti-rabbit Ig G, HRP-goat anti-mouse Ig G (1: 5000, Cell Signaling Technology, USA).

Cells were harvested and homogenized in the lysis buffer on ice using the proteo JET mammalian cell lysis reagent (Fermentas Life sciences, Israel). Protein concentration was determined using the Bio-Rad kit (Bio-Rad, Hercules, CA). The membranes with selected proteins were incubated at 4°C overnight with primary antibody against IGF1R (Cat #PA5-85986, Thermo Fisher Scientific, Inc.), MAPK1 (Cat #13-8600, Thermo Fisher Scientific, Inc.), MAPK3 (Cat #PA5-29636, Thermo Fisher Scientific, Inc.) and β-actin(Cat #MA5-15739, Thermo Fisher Scientific, Inc.), and then with the corresponding secondary antibody for 1h at room temperature. Immunofluorescence signals were detected using the ECL kit (Cell Signaling Technology, Inc.) and quantified using on the Gel Logic 2200 PRO Imaging System (Kodak, Rochester, NY, USA).

The collected cells were washed with PBS and lysed in RIPA lysis buffer (Beyotime) containing 2% protease inhibitor for 30 min. The protein concentration was determined using the BCA kit (Beyotime). Specifically, 20 μg of protein was boiled with 1 × loading buffer, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to polyvinylidene fluoride (PVDF) membranes. Following centrifugation at 12,000 × g for 20 min, the separated protein was injected into 10% SDS-PAGE and then transferred to PVDF membranes. After being blocked with 5% skim milk for 1–3 h, the membranes were incubated overnight with the primary antibodies [Runx2, ab76956, 1:1000; osteocalcin (OCN), ab93876, 1:1000; TLR4, ab22048, 1:1000; P65, ab32536, 1:1000; p-P65, ab76302, 1:1000; Abcam, Cambridge, UK] that were used for protein detection. The membranes were subsequently incubated with secondary antibody solution for 1 h after being washed with PBS. Again, the membranes were subjected to three rinses with PBS. The protein bands were visualized using an enhanced chemiluminescence (ECL) kit (Cell Signaling Technology) in a gel imager and underwent grayscale measurement with the help of Image J software. The relative expression level of the target protein was analyzed with β-actin as the internal control.

Western blotting was performed as previously described^{22 (link)}. Briefly, mesangial cells were lysed with RIPA lysis buffer in the presence of protease inhibitor cocktail (Merck, Darmstadt, Germany). Cell debris were removed by centrifugation at 12,000 rpm for 12 min. The protein content was determined with BCA kit and separated by 8–10% SDS-PAGE, and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were blocked in 5% (w/v) non-fat dry milk in Tris-buffered saline (TBS) and 0.1% (v/v) Tween 20 for 1 h at room temperature, and then incubated with the following primary antibodies at 4 °C overnight: BIRC6 (1:500) (Abcam, Cambridge, MA, USA); Ubiquitin, Bcl-2, Bax, cytochrome c, Cox IV, caspase-3 and caspase-9 (1:1000) (Cell Signaling Technology, Danvers, MA, USA); puma, Fas (1:500), p53, p21 and GAPDH (1:1000) (Santa Cruz Biotechnology, Paso Robles, CA, USA). After incubation of appropriate secondary horseradish peroxidase-conjugated antibodies including HRP-conjugated anti-rabbit or anti-mouse (1:1000; Cell Signaling Technology) for 1 h, blots were visualized with ECL kit and quantified with Image-Pro Plus 5.0 software.