Analyzing Cellular Protein Expression via SDS-PAGE and Western Blotting

SDS polyacrylamide gel electrophoresis and western blotting of total cellular extracts were performed as described previously [20 (link)]. The following primary antibodies were used: rat monoclonal anti-mouse GIMAP6 (generated in-house—either MAC 436 alone or a mixture of four different monoclonal antibodies MAC431, MAC432, MAC434, MAC436); mouse monoclonal anti-β-actin (Sigma); rat monoclonal anti-phosphoSQSTM1 (Ser403) (MBL); mouse monoclonal anti-SQSTM1 (Abnova); rabbit polyclonal anti-MAP1LC3B; rabbit monoclonal anti-phosphoTBK1 (Ser172); rabbit monoclonal anti-TBK1 (all from Cell Signalling Technologies). Rat monoclonal antibodies to other mouse GIMAP proteins were also generated in-house (GIMAP1 –MAC420; GIMAP4 –MAC415; GIMAP5 –MAC421; GIMAP7 –MAC448; GIMAP8 –MAC443; GIMAP9 –MAC433). Horseradish peroxidase-conjugated anti-mouse, anti-rat and anti-rabbit IgGs were purchased from Cell Signalling Technologies. Western blots were routinely developed using Immobilon Western reagents (Millipore Inc.), except for blots of actin when Supersignal West Pico Chemiluminescent Reagent (Thermo Scientific) was used. Blots were visualised using a G-box system (Syngene). Where required, band intensities were quantified using GeneTools software (Syngene).