Mouse monoclonal igg1

Cx43-hUCSCs were cultured on glass cover slips in six well plates and grown to 80% confluence. Cells were washed with PBS and fixed with 40 mg/L paraformaldehyde (Sigma, USA) for 25 min, then blocked with 10% goat serum albumin (Sigma, USA) for 20 min before staining. A drop of DAPI staining solution (Lifetechnologies, USA, 1 mg/ml) was added to stain the cells for 5 min. The slips of cells were washed with PBS, and a drop of 1:100 diluted mouse anti-human Cx43 monoclonal antibody (Abcam, USA) was added and incubated for 24 h at 4°C. Samples were warmed to room temperature for 1 h, washed with PBS, 1 drop of 1:100 diluted FITC-labeled goat anti-mouse IgG (Abcam, USA) was added and incubated for 1 h at room temperature. Mouse monoclonal IgG1 (Abcam, USA) was used as isotype control as instructed by manufactures. After washing with PBS, the slips were sealed using 60% buffering glycerol (Sigma, USA) and imaged by a laser confocal microscope (Leica, Germany) with a fast scanning mode.

After treatment with HI for 24 h, samples were analyzed for p53, PSMD11, Noxa, Mcl-1, Bax, Bcl-2 protein expression. Additionally, PSMD11 expression was studied in cells treated for 24 h with bortezomib or MG132. Briefly, 1 × 10⁶ cells were fixed and permeabilized and then incubated with the following antibodies: p53 [20 µL, Alexa Fluor® 488 Mouse anti-p53 (pS37), BD Biosciences (Franklin Lakes, NJ, USA)], PSMD11 [1:100, mouse monoclonal (AT1F4) anti-PSMD11, Abcam (Cambridge, United Kingdom)], Noxa [2:100, mouse monoclonal antibody (1144C307) anti-Noxa, Abcam], Mcl-1 (1:100, mouse monoclonal IgG1, Abcam), Bax [1:100, mouse monoclonal IgG1 (sc-20067), Santa Cruz Biotechnology (Santa Cruz, CA, USA)] Bcl-2 [1:100, mouse monoclonal IgG1 (sc-509), Santa Cruz Biotechnology]. The samples were washed and incubated, except p53, with the secondary fluorescein isothiocyanate (FITC) anti-mouse IgG (1:100, Sigma), and analyzed via flow cytometry. Mean fluorescence intensity values were calculated.