Rabbit monoclonal anti gapdh

The total protein was extracted from the cultured cells and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) and transferred onto a PVDF membrane (Bio-rad, United States). The membrane was incubated with rabbit monoclonal anti-BMP2 (1:1000; Proteintech, United States), rabbit monoclonal anti-BMP4 (1:1000; Affinity Biosciences, United States), rabbit monoclonal anti-DMP1 (1:1000; Abcam, United Kingdom), rabbit monoclonal anti-ALP (1:1000; Abcam, United Kingdom), mouse monoclonal anti-RUNX2 (1:1000; Abcam, United Kingdom), rabbit monoclonal anti-TGFβ (1:1000; Abcam, United Kingdom), rabbit monoclonal anti-DSPP (1:1000; Affinity Biosciences, United States), rabbit monoclonal anti-MSX2 (1:1000; Bioss, CHN), and rabbit monoclonal anti-GAPDH (1:1000; Abcam, United Kingdom) overnight at 4°C. Proteins were visualized using HRP-conjugated donkey anti-rabbit IgG (1:20000; BBI Life Sciences) and HRP-conjugated donkey anti-mouse IgG (1:20000; BBI Life Sciences) secondary antibody. The membrane was scanned on an Odyssey V3.0 scanner (Li-cor).

Lung tissues or cell pellets were lysed in RIPA buffer containing proteinase inhibitor. Equal amounts of protein (20 μg) were loaded on 8–10% SDS-PAGE gels and then electro-transferred onto PVDF membranes (Millipore Corp). The membranes were then blocked with BSA and incubated with the indicated primary antibody (1:3,000) overnight at 4 °C. Subsequently, the membranes were incubated with a secondary antibody (1:3,000, Abcam, anti-rabbit IgG). Then, the protein level on the blot was detected using the Western Bright ECL kit (Bio-Rad Laboratories). Equal loading of the samples was validated by the detection of GAPDH. The following antibodies against the target proteins in the study were used: rabbit monoclonal anti-FAK (Abcam), rabbit polyclonal anti-phospho-FAK (at Tyr397, Cell Signalling), mouse monoclonal anti-phosphated-Akt (at Ser 473, Santa cruz), and rabbit monoclonal anti-GAPDH (Abcam).

Cells were lysed in lysis buffer (Beyotime Biotechnology) that contained a phosphatase inhibitor cocktail. The supernatant was collected by centrifugation, and the protein concentration was determined with the BCA Protein Assay Kit (Beyotime Biotechnology). Equal amounts of total protein were loaded onto sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gels and resolved by electrophoresis. The separated proteins were transferred to a polyvinylidene fluoride membrane followed by incubation with 5% non-fat milk solution for 1 h at room temperature. Next, the membrane was probed with primary antibodies against the target proteins at 4 °C overnight. The antibodies were rabbit polyclonal anti-SOX30 (Invitrogen; #PA5–40508), rabbit monoclonal anti-GAPDH (Abcam; #ab9485), and rabbit monoclonal anti-active β-catenin (Cell Signaling Technology; #19807). Subsequently, the membrane was incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Abcam; #ab205718) for 1 h at room temperature. Finally, an enhanced chemiluminescence kit (Millipore) was used to visualize the protein bands. Bands of interest were quantified using Image-Pro Plus 6.0 software.

Proteins of mouse retina tissue were extracted with a protein extraction solution assay kit (RAPI, Sigma). Nanodrop (Thermo) was used to measure protein concentration. Equal amounts of proteins were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) resolving gel and 5% stacking gel and transferred onto polyvinylidene fluoride (PVDF) membranes. PVDF membranes were blocked with skim milk (BD) in Tris-buffered saline (TBS) for 1 h at room temperature. Following this, they were incubated with primary antibodies at 4 °C overnight (rabbit monoclonal anti-ZO-1, 1:500, Abcam; mouse monoclonal anti-RPE65, 1:500 Abcam; mouse polyclonal anti-Nestin, 1:500 Santa Cruz; rabbit monoclonal anti-α-SMA, 1:1000, Abcam; mouse polyclonal anti-CD68, 1:1000, Proteintech; rabbit anti-Bax, 1:1000, Abcam; rabbit monoclonal anti-GAPDH, 1:1000, Abcam). This was followed by PVDF membranes being washed with TBS-T (Tween 1:1000 dilution in TBS buffer) three times followed by incubation with secondary antibodies (HRP-goat anti-rabbit IgG, 1:2000, Bioss; HRP-goat anti-mouse IgG, 1:2000, Bioss) for 1 h at room temperature. After incubation, PVDF membranes were washed three times with PBS. Protein signals were detected with SuperSignal™ West Femto Maximum sensitivity substrate (Thermo Fisher Scientific) and imaged using the chemiluminescence system (Bio-Rad).