Anti myc tag antibody agarose

In this study, the following antibodies were used. Mouse primary antibodies: Myc tag, Drp1, OPA1, cytochrome c, and PARP (BD Biosciences); GAPDH (Santa Cruz); Mfn1, Mfn2, and Miro1 (Abcam); GFP and GST (Invitrogen). Rabbit primary antibodies: Dyn2 (Abcam); hFis1 (Atlas Antibodies); Myc tag (Sigma‐Aldrich); LC3B (Cell Signaling); and MTGM (Romo1) (Zhao et al, 2009). Secondary antibodies: DyLight 488‐, 594‐, and 649‐conjugated anti‐mouse and anti‐rabbit IgG antibodies (Vector Laboratories) for immunofluorescence and peroxidase‐conjugated anti‐mouse and anti‐rabbit IgG antibodies (GE healthcare) for Western blot. Alexa Fluor™ 488 Phalloidin (Invitrogen) was used for visualization of F‐actin by fluorescence. Dynabeads^® Protein G (Invitrogen) and anti‐Myc tag antibody agarose (Abcam) and anti‐GFP tag antibody agarose (MBL) were used for immunoprecipitation. The MitoTracker Red CMXRos (Thermo Fisher) was used for labeling mitochondria. Polyethylene glycol (PEG) 1500 and cycloheximide (Sigma) were used in cell fusion assay. Latrunculin B (LatB) (Sigma‐Aldrich) was used for inhibiting actin polymerization as previously described (Wakatsuki et al, 2001; Zackroff & Hufnagel, 2002; Korobova et al, 2013).

HEK 293T cells were transfected with siRNA via reverse transfection using Dharmafect 1 at a final concentration of 50 nm and seeded in regular 6-well–plates at a density of 1 × 10⁶ cells/well. After 24 h incubation, cells were transiently transfected the Myc-tagged KRAS^G12D plasmid using the Lipo6000 Transfection Reagent (Beyotime, Shanghai, China, catalog number C0526) per the manufacturer's protocol and further cultured for 48 h. Cells were lysed in Pierce IP Lysis Buffer supplemented with protease and phosphatase inhibitors (Roche), and lysates were further incubated with 10 μg of anti-Myc tag antibody (agarose) (Abcam, Cambridge, England, UK, catalog number ab1253) or IgG-agarose beads (Abcam, catalog number ab104155) for 8 h at 4 °C. After washing the agarose beads three times with Pierce IP Lysis Buffer, the Myc-tagged-KRAS bound to agarose beads was released by the addition of SDS lysis buffer. Finally, samples were subjected to Western blotting analysis as previously described and blots were probed using an anti-phosphotyrosine antibody (Cell Signaling Technology, catalog number 9411, 1:1000) or anti-Myc antibody (Cell Signaling Technology, 1:1000).