Anti sik2

Antibodies were raised against peptides encompassing amino acids 577–592 or 597–612 of human SIK1 (S306D, bleed 3), amino acids 272–445 of mouse SIK2 (S567D, bleed 3) and amino acids 926–1038 of mouse SIK3 (S373D, bleed 3) in sheep and affinity purified by the antibody production team at MRC-PPU Reagents and Services (https://mrcppureagents.dundee.ac.uk). The following antibodies were used for immunoblotting: horseradish peroxidase-conjugated secondary antibodies (Pierce), anti-HA (Roche), anti-CRTC3 (Abcam), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH), anti-nitric oxide synthase (NOS), anti-SIK2, anti-signal transducer and activator of transcription (STAT) 3, anti-pY705-STAT3 and anti-pS246-HDAC4/pS259-HDAC5/pS155-HDAC7 (Cell Signalling Technology).

Cells were lysed in 0.5 mL of lysis buffer, followed by at 14 000 g for 10 min at 4 °C. Lysates were then incubated with 5 μg of anti‐SIK2 antibody (Cell Signaling Technology) for 4 h, followed by centrifugation at 4 °C in the presence of 5 μL of prewashed protein‐A/G beads (Absin Bioscience, Shanghai, China). The beads were collected using a magnet and washed thrice with 1 mL of wash buffer. The immune‐precipitates were analyzed using SDS/PAGE and the proteins were captured using anti‐SIK2 (Cell Signaling Technology) and anti‐MYLK antibodies (Santa Cruz, Carlsbad, CA, USA).