Donkey anti mouse igg h l alexa fluor 647

Antibodies against XBP1s and Ac-K(Acetylated-Lysine) were obtained from Cell Signaling Technology (Danvers, MA, USA), and anti-SIRT3 antibodies were obtained from Proteintech (Wuhan, China) and Everest Biotech (Oxfordshire, UK). Donkey Anti-Rabbt IgG H&L (Alexa Fluor 488) and Donkey Anti-Mouse IgG H&L (Alexa Fluor 647) were obtained from Abcam (Cambridge, MA, USA). Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was purchased from Boster Bio (Wuhan, China). FITC anti-human CD3 Antibody, APC anti-human CD14 Antibody were obtained from BioLegend(San Diego, CA). The IMQ powder, MKC8866, 3-TYP and Honokiol were from MedChemExpress (Princeton, NJ, USA). IMQ cream was obtained from Sichuan Med-Shine Pharmaceutical Co., Ltd. (Sichuan, China).

At 24 h after transfection, HeLa cells were treated with or without 250 μM sodium arsenite for 30 min. HeLa cells on coverslips were washed twice in PBS and fixed for 15 min in 4% paraformaldehyde and then permeabilized with 0.5% Triton X-100. Coverslips were washed three times in PBS, then blocked with 3% BSA in TBST (Tris-buffered saline supplemented with 0.05% Tween 20) for 30 min. Cells were incubated with primary antibodies at room temperature for 1 h, and then with Alexa-Fluor-conjugated secondary antibodies and Hoechst 33342 (ThermoFisher) at room temperature for 30 min. Coverslips were mounted on glass slides with ProLong Gold antifade solutions (Molecular Probes). Mouse monoclonal anti-poly(ADP-ribose) polymer [10H] (1:50; Abcam) and rabbit monoclonal anti-HA [C29F4] (1:500, Cell Signaling) were used as primary antibodies. Secondary antibodies were donkey anti-mouse IgG H&L Alexa Fluor 647 (1:500; Abcam) and donkey anti-rabbit IgG H&L Alexa Fluor 488 (1:500; Abcam).
Microscopy was performed by using an LSM 880 confocal laser scanning microscope (Zeiss) with a 63× oil immersion objective (NA 1.4) and Zen imaging software (Zeiss). Images were collected simultaneously using the 405, 488 and 633 nm excitation laser lines.