Ab5594

Anti-A1AR antibody (ab82477), anti-active caspase-3 (ab2302), Chk pAb to albumin (ab106582), Rb pAb to MAPKAP2 (ab131531), Rb pAb to phosphor MAPKAP2 (ab63378), Ms mAb to Hsp27 (ab2790), Rb pAb to phosphor Hsp27 (ab5594), Rb mAb to NeuN (ab177487), anti-ERK1 (phospho T202) + ERK2 (phospho T185) antibody (ab201015), anti-ERK1 + ERK2 antibody (ab17942), anti-JNK1 (phospho T183) antibody (ab47337), anti-JNK1 antibody (ab110724), anti-GFAP antibody (ab10062), and Ms mAb to NeuN (ab104224) were purchased from abcam. Anti-A2aAR antibody (sc-32261), anti-A2bAR antibody (sc-28996), anti-A3AR antibody (sc-13938), β-actin (sc-47778), and GAPDH (sc-365062) were purchased from Santa Cruz. Rb pAb to P38, phosphor P38 was purchased from Cell Signaling.

The protein levels of Hsp27, p-Hsp27, HMGN5, E-cadherin, Vimentin, STAT3, and p-STAT3 were determined by immunoblotting. After lysing the target cells with RIPA buffer supplemented with 1% PMSF, we extracted the proteins and determined the protein concentration using the bicinchoninic acid (BCA) protein assay kit (Beyotime Institute of Biotechnology, China). An SDS-PAGE minigel was used for protein separation and the proteins were then transferred onto a PVDF membrane, which was then probed with the following antibodies (dilution 1:1000) : anti-Hsp27 (ab2790, Abcam), anti-p-Hsp27 (ab5594, Abcam), anti-HMGN5 (ab56031, Abcam), anti-E-cadherin (ab1416, Abcam), anti-Vimentin (ab8978, Abcam), anti-STAT3 (ab68153, Abcam), anti-p-STAT3 (ab76315, Tyr705, Abcam), and anti-GAPDH (ab8245, Abcam) at 4°C overnight followed by the incubation with the HRP-conjugated secondary antibody (dilution 1:5000). GAPDH was used as a reference protein. An ECL substrates (Millipore, MA, USA) was used to visualize the signals and ImageJ software (NIH) was used to perform the band intensity analysis.

The protein levels of HSP27, pHSP27, and G6PD in infarcted cortical tissue from each group were determined by immunoblotting according to a previously described method [6 (link)]. Five percent bovine serum albumin (BSA, Nacalai Tesque) was used for blocking, and primary antibodies were diluted in Can Get Signal (TOYOBO) at 4 °C overnight. The following antibodies were used: rabbit polyclonal antibodies against G6PD (1:1000, #8866, Cell Signaling Technologies), HSP27 (1:1000, #2442, Cell Signaling Technologies), and pHSP27 (S85) (1:4000, ab5594, Abcam) and a mouse monoclonal antibody against β-actin (1:4000, AM4302, Thermo Fisher Scientific). The blots were quantified using densitometric analysis performed with NIH ImageJ software (ver. 1.51, https://imagej.nih.gov/ij/). β-actin was used as a loading control. The experiments were repeated two times.