Ab32988

2 × 10⁶ OCI-AML2 cells were pelleted and frozen at −80 degrees. For the detection of POLRMT, cells were lysed in RIPA lysis buffer and proteins were separated on a 7.5% PAGE minigel (Bio-Rad Laboratories). For mitochondrial proteins, cells were lysed in 1.5% n-beta-maltoside (Sigma) dissolved in PBS. Lysates were supplemented with Halt protease inhibitors (Thermo Scientific) and 0.5 mM EDTA. For mitochondrial proteins, separation was performed on a 12% PAGE gel. Semi-dry transfer was performed (Trans-Blot SD, Bio-Rad) onto PVDF membranes. Primary antibodies and concentrations were: POLRMT, Abcam ab32988, 1:250; OXPHOS cocktail, Abcam ab110411, 1:1,000 (contains antibodies against complex I, NDUFB8, complex II, SDHB, complex III, UQCRC2, complex IV, COXII, complex V, ATP5A), and alpha-tubulin, 1:1000.

Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The blots were probed with primary antibodies to known mitochondrial RNA-interacting proteins; LRPPRC (Abcam, ab21864, 1:1000), POLRMT (Abcam, ab32988, 1:5000), SUV3 (kind gift of Dr. Roman Szczesny, 1:5000) and GRSF1 (Sigma, HPA036985, 1:5000), bound by HRP-conjugated secondary antibodies (Vector Laboratories, 1:10000) and detected by ECL in a ChemiDoc instrument (Biorad).