Anti rabbit igg conjugated to alexa fluor 488

HNSCC cells were plated on 18-mm cover glasses until they adhered to the surface. The cells were fixed and permeabilized with 0.1% Triton X-100. Then, they were blocked by 5% bovine serum albumin (BSA) and incubated with primary antibodies against PSMD14 and SOX2 at 4°C overnight. The proteins were visualized by incubation with anti-rabbit IgG conjugated to Alexa Fluor 488 or anti-mouse IgG conjugated to Alexa Fluor 594 (Cell Signaling Technology) for 1 hour at room temperature, and the nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific) for another 10 min. All images were obtained by Iamger.Z2 (Zeiss, Oberkochen, Germany).

HNSCC cells were seeded onto 18 mm cover glass and incubated overnight at 37 °C. After methanol immobilization, the cells were permeabilized with 0.2% Triton X-100. Cells were then sealed with 2% BSA and incubated with primary antibodies at 4 °C for 14 h. Cells were then incubated with anti-rabbit IgG conjugated to Alexa Fluor 488 or anti-mouse IgG conjugated to Alexa Fluor 594 (Cell Signaling Technology) for 60 min at room temperature, and the nuclei were stained with 4,6-diamidino-2- phenylindole (DAPI; Thermo Fisher Scientific) for another 10 min. All images were captured using an Axio Imager Z2 microscope (Zeiss, Oberkochen, Germany).

rhIGFBP-3 was acquired from Sino Biological (Chesterbrook, PA, USA). The lyophilized protein was re-suspended using ultrapure water. Antibodies used for immunoblotting and immunofluorescence included: a rabbit polyclonal anti-IGF-1Rβ #3027, a rabbit monoclonal anti-mitofusion-2 #9482, a rabbit monoclonal anti-mitofusion-1 #14739, a rabbit monoclonal anti-OPA1 #67589, a rabbit monoclonal anti-COX IV #4850, a rabbit polyclonal anti-VDAC1 #4661 (Cell Signaling, Danvers, MA, USA); a mouse monoclonal anti-GAPDH #sc-66163, a mouse monoclonal anti-β-actin #sc-47778 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); and a mouse monoclonal anti-puromycin #MABE343 (EMB Millipore, Burlington, MA, USA). Secondary antibodies used for immunofluorescence were anti-rabbit IgG conjugated to Alexa Fluor 488 and anti-mouse IgG conjugated to Alexa Fluor 555 (Cell Signaling, Danvers, MA, USA). For immunoblotting, secondary antibodies were goat anti-rabbit IgG conjugated to horseradish peroxidase (HRP) #170-6515 and goat anti-mouse conjugated to HRP #170-6516 (Bio-rad, Hercules, CA, USA).