Protein lysates were prepared using RIPA buffer containing 1 mM PMSF, 1 mM sodium orthovanadate, 1 mM NaF, and 30 µL/mL aprotinin. The proteins (20–30 µg) were resolved in SDS-PAGE, electro-transferred onto nitrocellulose membranes (Hybond-ECL, GE Healthcare) and blocked with 5% BSA. Primary antibodies included NRF2 (D1Z9C), BiP/GRP78 (C50B12), IRE1α (14C10), CHOP (L63F7), Calnexin (C5C9) and PERK (D11A8) from Cell Signaling Technologies; p-IRE1α (Ser724; NB100-2323) from Novus Biologicals; GCLC (Ab41463), beta-actin (Ab8227), TBP (ab818) and ATF6 (Ab37149) from Abcam; p-PERK (Thr 981, sc-32577), NQO1 (C-19), and ATF3 (C19) from Santa Cruz. After secondary antibody incubation (1:3000 in TBS-T, 2 h), the proteins were detected using Lumiglo substrate (Cell Signaling Technology, CA) and X-ray films. Immunoblot images are representative of three independent experiments.
P ire1α ser724 nb100 2323
Manufactured by Novus Biologicals
P-IRE1α (Ser724; NB100-2323) is a phospho-specific antibody that recognizes the serine 724 phosphorylation site of the IRE1α (Inositol-Requiring Enzyme 1α) protein. IRE1α is a key sensor of the unfolded protein response (UPR) pathway.
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Lab products found in correlation
Lumiglo substrate, by Cell Signaling Technology (1 mentions)
Hybond ecl, by GE Healthcare (1 mentions)
Atf3 c19, by Santa Cruz Biotechnology (1 mentions)
Hybond, by Cytiva (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
Goat anti rabbit igg h l secondary antibody, by Thermo Fisher Scientific (1 mentions)
Alexa488 conjugated anti mouse, by Jackson ImmunoResearch (1 mentions)
Lc3 l8918, by Merck Group (1 mentions)
Ire1α, by Cell Signaling Technology (1 mentions)
2 protocols using p ire1α ser724 nb100 2323
1
Western Blot Analysis of UPR Proteins
2
Antibody Sources and Reagents for ER Stress
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Antibodies were purchased from the following sources: CCPG1 (ab219854) and KDEL (ab12223) were from Abcam; RTN3 (PA2256) was from Boster Biological Technology; ATF4 (#11815), ATF6 (#65880), Bcl-2 (#2870), Beclin1 (#3738), CHOP (#5554), COX IV (#4844), GAPDH (#2118), IRE1α (#3294), p-PERK (Thr980, #3179), and PERK (#3192) were from Cell Signaling Technology; FAM134B (NBP2-55248) and p-IRE1α (Ser724, NB100-2323) were from Novus Biologicals; LC3 (L8918) and p62 (P0067) were from Sigma. The secondary antibodies were purchased from the following sources: Goat anti-rabbit IgG (H + L) secondary antibody (31460) was from Thermo Fisher Scientific and Alexa488-conjugated anti-mouse (715–545–150) was from Jackson ImmunoResearch. Reagents were purchased from the following source: Bafilomycin A1 (B1793), carbonyl cyanide 3-chlorophenylhydrazone (CCCP; C2759), and sodium phenylbutyrate (4PBA; SML0309) were from Sigma.
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