Xp04205box

Whole cell lysates were prepared by scraping cells directly into Ripa buffer (50 mM Tris-HCl, 1% NP-40, 0.5% Na-Deoxycholate, 0.1% SDS, 150 mM NaCl, 2 mM EDTA) supplemented with protease and phosphatase inhibitors (Sigma Aldrich P8340, Roche Molecular 04906845001), incubated on ice for 45 min with vortexing every 5 min, and then centrifuged at 16,000 x g for 10 min at 4°C to remove insoluble material. Supernatant was saved as whole cell lysate (WCL). WCL, PMS, and/or crude mitochondrial fractions were normalized for total protein content via BCA Assay (Thermo Scientific 23225). Samples were resolved by SDS-PAGE on Tris-glycine gels (Invitrogen XP04205BOX) and transferred to nitrocellulose or PVDF membranes. Immunoblotting was performed using the indicated primary antibodies which are listed in the key resources table according to the manufacturers’ recommendations, and analyzed by Licor Odyssey.

Full-length human FAM46A/FAM46C with a FLAG-tag and BCCIPα/BCCIPβ with a Myc-tag were cloned into the pRK5 vector (556104, BD PharMingen). The plasmids were transfected into HEK293T cells with Lipofectamine 3000 (L3000001, Invitrogen) according to the manufacturer’s instruction. Cells were harvested 36 hours after transfection and lysed in the lysis buffer containing 50 mM tris (pH 8.0), 150 mM NaCl, 0.1% NP-40, 1 mM DTT, and protease inhibitor (78442, Sigma-Aldrich). Lysates were cleared by centrifugation at 15,000 rpm for 10 min at 4°C. anti-FLAG beads (A2220, Sigma-Aldrich) were added to the supernatant, and the mixture was rotated at 4°C for 1 hour. The beads were washed with a lysis buffer three times. Proteins remaining on the beads were resolved with 4 to 20% gradient SDS-PAGE (XP04205BOX, Invitrogen) and detected by Western blot using anti-FLAG (F7425, Sigma-Aldrich) and anti-Myc antibodies (2278, Cell Signaling Technology).

Whole cell lysates were prepared by scraping cells directly into RIPA buffer (or adding RIPA buffer to Jurkat cell pellets) supplemented with protease and phosphatase inhibitors (Sigma-Aldrich P8340, Roche Molecular 04906845001), incubated on ice for 30 min with vortexing every 10 min, and then spun at 16,000 × g for 10 min at 4°C to remove insoluble material. Supernatant was saved as lysate and concentrations were normalized for total protein content after measuring with a BCA (Thermo Scientific 23225). Samples were resolved by SDS-PAGE or Tris-glycine gels (Invitrogen XP04205BOX) and transferred to nitrocellulose or PVDF (extraction assay) membranes. Immunoblotting was performed using the indicated primary antibodies which are listed in the Key resources table according to the manufacturers’ recommendations, and analyzed by Licor Odyssey or Azure C500 (extraction assay). Note that the detector for the Azure C500 has several columns of pixels which appear to be non-functional. This gives the appearance of thin vertical white lines in some images. This can be readily viewed in raw data files by over-adjusting the contrast.