Ab32559

Proteins were extracted from tissue homogenates. For Western blot analysis, protein samples were fractionated in 4–20% SurePAGE™ Precast Gels (M00657, GenScript Biotech Corporation, China) and transferred to nitrocellulose membranes. The analysis was performed with anti-CCN1 (ab228592, Abcam, UK), anti-CDC42 (2466T, Cell Signaling Technology, USA), anti-Claudin5 (4C3C2, Invitrogen, USA), anti-COX6c (ab150422, Abcam, UK), anti-CREB1 (R23983, ZEN- BIOSCIENCE, China), anti-HIF1α (ab179483, Abcam, UK), anti-NDUFα1 (15561-1-AP, Proteintech Group, USA), anti- SEPN1 (55333-1-AP, Proteintech Group, USA), anti-SHP1 (ab32559, Abcam, UK, anti-VEGFa (NB100-664, Novus Biologicals, USA), and anti-β-tubulin (2146S, Cell Signaling Technology, USA) antibodies. Immunoassays were performed using enhanced chemiluminescence (17046; ZEN-BIOSCIENCE, China), in accordance with the manufacturer’s instructions. Protein levels were determined by densitometric scanning of protein bands. Six retinas were used in each group, and the results for each retina were averaged from three replicates.

Proteins were obtained using radioimmunoprecipitation assay (RIPA) buffer to dissolve the cells. The quantification was tested using bicinchoninic acid Protein Assay Kit (Boster Biological Company, Ltd., Wuhan, China). All protein samples were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The target strip was transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Burlington, MA, USA) via electrophoresis. The PVDF membrane was blocked in 5% nonfat milk. The protein bands were incubated with specific primary antibodies as follows: HNRNPH1 (1:2,000) (ab 154894, Abcam, CA, USA), PTPN6 (1:1,000) (ab 32559, Abcam, CA, USA), AKT (1:1,000) (ab 38449, Abcam, CA, USA), p-AKT (1:1,000) (ab 8805, Abcam, CA, USA), and β-ACTIN (1:8,000; Abways Technology, New York, NY, USA; AB0035). Moreover, the PVDF membrane was incubated in a goat-anti-rabbit secondary antibody (1:10,000, Boster Biological Company, Ltd., Wuhan, China) overnight. Images of protein quantification were captured by BioSpectrum Imaging System (UVP, LLC, Upland, CA, USA).