Rabbit anti human monoclonal antibodies

siRNAs for Fas, TNF-R1 or FADD were purchased from Qiagen (Valencia, CA). The siRNAs chosen had been functionally verified in human cells by the manufacturer. Their sequences are shown in Table 1. A negative control siRNA was not used in this study, because in a previous study we reported that Allstars negative control siRNA (Qiagen) had no effect on the response of K⁺ channels and activation of apoptotic mechanisms in HCLE cells exposed to UVB (Ubels et al., 2016 (link)).
Prior to transfection, 2.5 µL/mL siLentFect (BioRad, Hercules, CA) and 25 nM siRNA were mixed with Opti-MEM (Life Technologies, Carlsbad, CA) and incubated together for 20 min at room temperature. HCLE cells, which had been grown to 30–50% confluence in six-well plates, were transfected using the Opti-MEM mixture according to the manufacturer's protocol. Knockdown of proteins was confirmed by SDS-PAGE and western blotting using rabbit antihuman monoclonal antibodies (Cell Signaling Technology, Danvers, Massachusetts) and Odyssey IRDye800 goat anti-rabbit secondary antibodies (Li-Cor, Lincoln, NE). Blots were imaged and scanned with a Li-Cor Odyssey Infrared Imaging System.

Small interfering RNAs (siRNA) were purchased from Qiagen (Valencia, CA). HCLE cells were grown to 30–50% confluence and transfected with siRNAs to Fas (25 nM), caspase-8 (25 nM), Apaf-1 (50 nM), or All-Stars negative control siRNA (50 nM) using siLentFect (BioRad, Hercules, CA), according to manufacturers’ protocols. Optimal transfection conditions and siRNA concentrations were determined in preliminary experiments (data not shown). After transfection, cells were incubated for 72 hours. Knock-down of proteins was confirmed by SDS-PAGE and western blotting using appropriate rabbit anti-human monoclonal antibodies (Cell Signaling Technology, Danvers, MA) and Odyssey IRDye800 goat anti-rabbit secondary antibody (Li-Cor, Lincoln, NE). All control data in knockdown experiments are from cells transfected with the negative control siRNA. Blots were imaged and scanned with a Li-Cor Odyssey Infrared Imaging System.