Ab137093

Different treated HK2 cells, plated on glass coverslips in a 35-mm dish were fixed with 4% paraformaldehyde, permeabilised with 0.2% Triton X-100 and blocked with 10% bovine serum albumin (BSA). The cells were then incubated with primary antibodies: monoclonal mouse anti-human HIF-1α (ab1, Abcam, Cambridge, MA, USA) and monoclonal rabbit anti-human RAB22A antibodies (ab137093, Abcam); followed by incubation with secondary antibodies: Cy3-conjugated goat anti-mouse immunoglobulin G (IgG, GB21301, ServiceBio, Wuhan, China) and fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (GB22303, ServiceBio). DAPI, (G1012, ServiceBio) was used to stain nuclei. The samples were visualised using the Pannoramic 250 Flash series digital scanner and software (3DHistech, Budapest, Hungary).

Total protein was extracted using the RIPA lysis buffer (Beyotime), and was separated by SDS‐PAGE gels and then transferred onto the PVDF membrane (Merck Millipore). The membranes were blocked with 5% nonfat milk, and then incubated with primary antibodies overnight at 4°C and goat anti‐rabbit secondary antibody (ab6721; 1:5 000; Abcam) at room temperature for 1 h. Finally, images were developed with the Immuno Star LD (Wako Pure Chemical). The antibodies were listed as follows: anti‐RAB22A (ab137093; 1:1 000; Abcam), anti‐Bax (ab32503; 1:1000; Abcam), anti‐Bcl‐2 (ab32124; 1:500; Abcam), OCT4 (ab200834; 1:10000; Abcam), NANOG (ab109250; 1:2 000; Abcam), CD133 (ab222782; 1:2000; Abcam) and anti‐β‐actin (ab8227; 1:1 000; Abcam).

Total protein was extracted from the mouse brain tissues and NSCs using RIPA lysis buffer (Abcam) along with protease and phosphatase inhibitors (Thermo Fisher Scientific). The protein content was determined using a BCA Protein Assay Kit (Thermo Fisher Scientific). Similar quantities of proteins were separated via sodium-dodecyl sulfate gel electrophoresis (Millipore, MA, United States) and then transferred to polyvinylidene fluoride membranes. The membranes were incubated overnight and at 4 °C with primary antibodies (all from Abcam, Cambridge, United Kingdom) against Rab22a (1:1000, ab137093), p65 (1:1000, ab32536), phospho (p)-p65 (1:1000, ab76302), Bax (1:1000, ab32503), Bcl-2 (1:2000, ab182858), and GAPDH (1:2000, ab181602). Next, the membranes were treated with HRP-conjugated secondary antibodies (1:3000, Abcam) for 1 h at 22 °C. Electrochemiluminescence Western Blotting Substrate (NIH, Bethesda, MD, United States) was used to visualize the protein bands, which were quantified using ImageJ software.