Anti ptpip51 20641 1 ap

We harvested the total protein from the ischemic penumbra 1 day after ischemia/reperfusion (I/R) using RIPA lysis buffer, then loaded the samples for SDS‐PAGE, then we used the PVDF membranes (Millipore) to transfer proteins. The PVDF membranes were blocked with 5% bovine serum albumin (BSA) solution for 1 h, then incubated overnight at 4°C with the following primary antibodies, anti‐VAPB (95339, Cell Signaling Technology), anti‐PTPIP51 (20641‐1‐AP, Proteintech), anti‐Mfn2 (12186‐1‐AP, Proteintech), anti‐β‐actin (1967, Cell Signaling Technology), anti‐Beclin‐1 (2738, Cell Signaling Technology), anti‐P62 (39749, Cell Signaling Technology), anti‐LC3I/II (12741, Cell Signaling Technology), anti‐PI3K (4257, Cell Signaling Technology), anti‐p‐PI3K (17366, Cell Signaling Technology), anti‐AKT (9272, Cell Signaling Technology), anti‐p‐AKT (3038, Cell Signaling Technology), anti‐mTOR (2972, Cell Signaling Technology) and anti‐p‐mTOR (2971, Cell Signaling Technology). The membranes were rinsed three times with phosphate‐buffered saline (PBS)/0.1% Tween, then incubated with secondary antibodies for 1 h. Images were observed using a Bio‐Rad ChemiDoc western blot analysis system, and protein expression levels were normalized to β‐actin with ImageJ.