Anti ptpip51

The brain tissues were collected in each group following perfusion with ice‐cold PBS 1 day after MCAO, and fixed with 4% paraformaldehyde for 48 h, then cut into slices. The slices were incubated in 0.3% Triton X for 10 min and blocked with 5% fetal bovine serum for 60 min at room temperature. The slices were incubated with the following primary antibodies overnight at 4°C after rinsing with PBS thrice, anti‐VAPB (66191, Proteintech), anti‐PTPIP51 (20641, Proteintech), and anti‐NeuN (94403, Cell Signaling Technology). The slices were washed thrice and then incubated with an Alexa 488‐conjuated antibody (ANT024S; Millipore, Billerica, MA) or Alexa 594‐conjugated antibody (ANT029S, Millipore, Billerica, MA) for 2 h at room temperature. DAPI was used to stain cell nuclei after washing with PBS. We randomly selected three brain sections from the ischemic region of each animal and observed VAPB, PTPIP51, NeuN immuno‐positive cells in the penumbra with an automated fluorescence microscope (Olympus, Japan). The number of positively stained cells was calculated and averaged with ImageJ. Statistical analysis was performed using five mice in each group.

Anti‐VDAC 1:1000 (Cell Signaling, 4661), anti‐PTPIP51 1:1000 (Proteintech, 20,641‐1‐AP), anti‐VAPB 1:1000 (Invitrogen, MA5‐24348), anti‐Grp75 1:5000 (Proteintech, 14,887‐1‐AP), anti‐Bap31 1:1000 (Enzo Life Sciences, ALX‐804‐601‐C100), anti‐Fis1 1:1000 (GeneTex, GTX111010), anti‐Tomm40 1:1000 (Novus Biologicals, NBP2‐94075), anti‐IP3R‐I/II/III 1:500 (Santa Cruz Biotechnology, sc‐377,518), anti‐H3 0.5 μg/ml (GenScript, A01502), anti‐Actin 1:200 (Santa Cruz Biotechnology, sc‐8432).

Dasatinib (Cayman Chemical Company, 11,498) is an inhibitor for non‐receptor tyrosine kinases such as cAbl and members of the Src family. Gefitinib (Cayman Chemical Company, 13,166) inhibits EGFR and EGFR‐associated kinases. Rp‐cAMPs (Enzo Life Sciences, BML‐CN135‐0001) is an inhibitor for cAMP‐dependent protein kinases such as PKA. All three kinase inhibitors have been shown to modulate PTPIP51 phosphorylation and the ability for PTPIP51 to interact with other proteins.²⁹ LUHMES were differentiated on glass coverslips coated in PLO and fibronectin and treated with Tat or PBS for 24 h as described above. Dasatinib (400 nM), Gefitinib (80 μM), and Rp‐cAMPs (40 μM) were added to the cells at the same time and allowed to incubate for 4 h. The cells were then washed in PBS and fixed in 4% formaldehyde in PBS for 20 min. PLA was conducted as described above using anti‐pTyr 1:350 (Cell Signaling, 9411) and anti‐PTPIP51 (Proteintech, 20,641‐1‐AP) primary antibodies. Images were taken on a Leica EL6000 DMI3000 confocal microscope and analyzed using ImageJ.