Parp h 250

The expression levels of proteins were determined by Western blotting as described earlier [26 (link)]. Immunoassays were performed with anti-p53 (DO-1), -p21 (C-19), -caspase 7 (C-18), -caspase 9 (H-83), -Bcl-2 (N-19), -Bax (N-20), -PARP (H-250) (Santa Cruz Biotechnology, Inc.), anti-cyclin B1, -caspase 3 (BD biosciences), anti-hnRNP-K (ImmuQuest), anti-pRb (ser-780), anti-actin (Chemicon International, CA) and anti-mortalin antibodies [33 (link)]. Quantitation of immunoblots was performed using the Image J software (National Institute of Health).

Antibodies directed against EphA2 (WB analysis for total EphA2), Chlamydia Hsp60, GP96, PARP H-250, Pan Cadherin, GAPDH were purchased from Santa Cruz biotechnology. Antibodies against N-terminal EphA2 (D4A2) for WB and IF studies, pEphA2 (phospho EphA2 Ser897), pERK, pAkt, pPI3K, p85-PI3K, total Akt and total ERK were from Cell Signaling technology. Antibodies against N-terminal EphA2 (FACS, WB and IF), EphB4, PDGFRβ and IgG (control) were from R&D systems. Antibody against PDI was bought from Thermo Scientific. Anti-β-Actin and anti-Flag antibodies were purchased from Sigma-Aldrich. Phalloidin was bought from Invitrogen. Draq5 was from BioStatus Limited, UK. Polyclonal serum against IncA (anti-rabbit) was obtained through Gramsch laboratories. Inhibitors for PI3 kinase (LY294002), MEK1/2 (UO126) and EphA2 (dasatinib) were bought from Cell Signaling. Recombinant human EphA2 (rhEphA2) and recombinant human Ephrin-A1 homodimer (rhEphrin-A1 fused with IgG1-Fc) were purchased from R&D systems. Recombinant human prohibitin-His (rhPHB) also possess a His tag and is a control for rhEphA2. As a control for rhEphrin-A1, recombinant IgG1-Fc was bought from Life Technologies.

Caspase-3 (CAS3) and poly (ADP-ribose) polymerase 1 (PARP1) levels were determined in aqueous humor and plasma samples from POAG and cataract patients by Western blot and immunoblotting procedures, as described by Chandra and Tang [42 ].
First, we analyzed the concentration of total proteins following a procedure similar to Lowry’s [43 (link)]. Then, 30 µg protein were loaded on a 4–12% Bis-Tris gel and proteins are separated by electrophoresis. After, we carried out the transfer to the nitrocellulose membrane and finally CAS3 and PARP1 were detected by immunoblotting using specific antibodies (PARP H-250, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA; Cleaved Caspase-3, Cell Signaling Technology, Danvers, MA, USA). The band optical density was determined and analyzed by Scion Image Analysis (Scion Corp., Frederick, MD, USA).