Ecl western blot detection system

SG tissues were ground to extract total protein. The total protein concentration was determined using the BCA Protein Assay Kit (Cwbio, China). Equal amounts of proteins were subjected to 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Membranes were blocked using 5% skimmed milk and subsequently incubated overnight at 4°C with anti-calmodulin (CALM) (Bioss Antibodies, China; 1:1,000), anti-tropomyosin 1 (TPM1) (Boster, China; 1:1,000), anti-tropomyosin 2 (TPM2) (Bioss Antibodies, China; 1:1,000), and anti-β-actin (TransGen Biotech, China; 1:5,000). Then, the membranes were incubated using secondary antibodies, including goat anti-Mouse IgG (H+L) HRP (Promega Corporation, USA; 1:5,000), or goat anti-rabbit IgG (H+L) HRP (Promega Corporation, USA; 1:5,000). Last, protein signals were assessed and analyzed using the ECL western blot detection system (Millipore, Billerica, MA, USA).

Total proteins were isolated from the renal tissues using the RIPA buffer, and protein concentrations were quantified using the BCA protein assay kit (Beyotime, Shanghai, China). Protein samples (35 μg/lane) were resolved via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred onto polyvinylidene difluoride membranes (Beyotime, Shanghai, China). The membranes were incubated overnight at 4° C with rabbit anti-mouse fibronectin (1:1000), mouse anti-mouse α-SMA (1:200), mouse anti-mouse E-cadherin (1:500), rabbit anti-mouse Sphk1 (1:1000) (All from Abcam, Cambridge, MA, USA) followed by incubation with horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG secondary antibodies (1:3000; ZSGB-BIO, Beijing, China). Immunoblots were visualized using the ECL Western blot Detection System (Millipore, Billerica, MA, USA). GAPDH was used as the loading control.

After whole cell protein extracts were prepared in ice-cold RIPA lysis buffer and quantified by BCA (bicinchoninic acid) protein assay, equivalent amounts of cell lysates were separated by 8–12% SDS polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride (PVDF) membrane, which was then blocked in 5% non-fat milk in PBST (1X Phosphate Buffered Saline Tween-20) and probed overnight at 4 °C with the primary antibodies against human CD133 antibody (1: 1000, orb18124, Biorbyt) and β-actin (Sigma-Aldrich Corp., St. Louis, MO, USA). Anti-mouse or anti-rabbit IgG conjugated to horseradish peroxidase was used as the secondary antibody for detection using an enhanced chemiluminescence (ECL) western blot detection system (Millipore, Bedford, MA, USA), and band intensities were quantified by densitometry (Digital Protein DNA Imagineware, Huntington Station, NY).

Total proteins were extracted from the thoracic aorta tissues using the radioimmunoprecipitation buffer lysis buffer (Roche, Mannheim, Germany). Total protein concentration was measured using the BCA Protein Assay Kit (Pierce, Rockford, USA) according to manufacturer's instructions. The same amount of total proteins were injected into the sample holes of 10% sodium dodecyl sulfonate- (SDS-) polyacrylamide gel electrophoresis (PAGE) and then electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After being blocked with 5% skim milk for 1 h, the membranes were incubated overnight at 4°C with anti-p38 (Affinity Bioscience, USA; AF6456, 1 : 1000), anti-p-p38 (Affinity Bioscience, USA; AF4001, 1 : 1000), anti-JNK (Abcam; ab179461, 1 : 1000), anti-p-JNK, (Abcam; ab124956, 1 : 5000), anti-ERK (Affinity Bioscience, USA; AF0155, 1 : 5000), anti-p-ERK (Affinity Bioscience, USA; AF1015, 1 : 2000), and anti-β-actin (Abcam; ab8226, 1 : 5000). The membranes were then incubated with secondary antibodies, including goat anti-mouse IgG-H&L (HRP) (Abcam; ab205719, 1 : 10000) and goat anti-rabbit IgG-H&L (HRP) (Abcam; ab205718, 1 : 10000). Protein signals were assessed using enhanced chemiluminescence (ECL) western blot detection system (Millipore, Billerica, MA, USA) and densitometric analysis.

After cells were transfected with the designated plasmids, cells were harvested into universal protein extraction lysis buffer (Cat. No. PP1801, Bioteke, Beijing, PR China) containing protease inhibitor cocktail (Cat. No. 04693116001, Roche, Basel, Switzerland). Extracted proteins were incubated 1–3 μg target antibody or IgG as negative control overnight, and followed with protein A+G agarose beads (Cat. No. P2012, Beyotime, Jiangsu, PR China) for 2 h. Precipitated proteins were subjected to SDS-PAGE, transferred to PVDF membrane (Cat. No. IPVH00010, Millipore, Massachusetts, USA), and detected with specific appropriate primary antibodies and horseradish peroxidase-conjugated secondary antibodies. Specific proteins were visualized using an enhanced chemiluminescence (ECL) Western blot detection system (Millipore).

The detailed procedure for western blotting has been described [44 (link)]. Western blotting used primary antibodies (diluted 1:1000) against the following proteins: AKT (sc-8312), phosphorylated AKT (sc-16646-R), ERK (sc-94), phosphorylated ERK (sc-7383), and vimentin (sc-6260); each antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against HIF-1α (Cell Signaling) and E-cadherin (cat. 610182, BD Biosciences, Franklin Lakes, NJ) were also used. Proteins separated by SDS-PAGE were transferred to a Hybond-C Extra membrane (GE Healthcare, Little Chalfont, UK) that was then subjected to western blotting with an appropriate primary antibody. Anti-mouse or anti-rabbit IgG conjugated to horseradish peroxidase was used as the secondary antibody for detection using an ECL western blot detection system (Millipore, Bedford, MA, USA), and band intensities were quantified by densitometry (Digital Protein DNA Imagineware, Huntington Station, NY).