Rabbit monoclonal anti tbk1

Samples were resolved by SDS–PAGE and transferred onto a 0.45-μm nitrocellulose membrane. Rabbit monoclonal anti-GAPDH (2118, 1:5,000; Cell Signaling), mouse monoclonal anti-IRF3 (50772, 1:100; Abcam), rabbit monoclonal anti-TBK1 (3504, 1:1,000; Cell Signaling), and rabbit monoclonal anti-STING (13647, 1:1,000; Cell Signaling) blots were blocked in 5% nonfat powdered milk dissolved in Tris-buffered saline containing 0.1% Tween (TBST). Rabbit monoclonal anti–phospho-IRF3 (Ser396 4947, 1:1,000; Cell Signaling), rabbit monoclonal anti–phospho-STING (Ser366 19781, 1:1,000; Cell Signaling), rabbit monoclonal anti–phospho-TBK1 (Ser172 5483, 1:1,000; Cell Signaling), and rabbit monoclonal anti-phospho-H3 (Ser10 3377, 1:10,000; Cell Signaling) blots were blocked in 100% Odyssey Blocking Buffer (927–40000; LI-COR). Goat antirabbit DyLight 680 (3568; Pierce), goat antimouse DyLight 680 (35518; Pierce), goat antirabbit DyLight 800 (535571; Pierce), and goat antimouse DyLight 800 (35521; Pierce) were used as secondary antibodies at 1:10,000 in either 50% Odyssey Blocking Buffer/TBST or 5% milk/TBST. Blots were imaged with the Licor Odyssey Infrared Imaging System. Band intensities were quantified by densitometry using ImageJ v1.52a (104 (link)).

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).