Flag tag m2

Cells were fixed with 4% Pfa, permeabilized with 0.2% Triton, blocked in 1% BSA and were stained with anti-PI(3)P (Echelon), LBPA (Echelon), Atg12 (Cell Signaling #2011), WIPI2 (Abcam), Flag-tag M2 (Sigma), and Myc-tag 9B11 (Cell Signaling) and imaged on upright and inverted confocal microscopes made by Zeiss (Göttingen, Germany) including Zeiss 510 and Zeiss 780. 40× and 63× oil objectives were used. For quantification, at least 30 transfected cells imaged from at least three separate experiments were examined and categorized as indicated (i.e. no rings vs. rings).

17β-estradiol (E2), 4-hydroxytamoxifen (OHT), and tunicamycin were purchased from Sigma-Aldrich. E2 and OHT were dissolved in ethanol, tunicamycin in dimethyl sulfoxide (DMSO). Primary antibodies were the following: against the FLAG tag (M2, Sigma Aldrich), α-tubulin (DM1A, Sigma Aldrich), histone H3 (ab1791, Abcam), pERK1/2 (E-4, Santa Cruz Biotech), ERK2 (C-14, Santa Cruz Biotech), GAPDH (ab9484, Abcam), ERα (HC-20, Santa Cruz Biotech), GPER (LS-A4272, LifeSpan Biosciences), vimentin (V9, Abcam), and E-cadherin (H-108, Santa Cruz Biotech).

Samples were lysed in RIPA buffer (Thermo Fisher Scientific) and centrifuged at 14,000 rpm at 4 °C for 20 min. Supernatants were diluted in 2X Laemmli sample buffer (Bio-Rad). Proteins were separated on 10 or 4–20% gradient Mini-PROTEAN TGX gels (Bio-Rad) and transferred to nitrocellulose blotting membranes (Bio-Rad). Membranes were probed with primary antibodies against FLAG tag (M2, Sigma), MCU (Sigma), MICU1 (Abcam, Cambridge, MA), cytochrome C (Cell Signaling, Danvers, MA), Cyclophilin D (Millipore, Billerica, MA), Tim23 (BD Biosciences, San Jose, CA), VDAC1 (Abcam), COX1 (Abcam), GAPDH (Thermo Fisher Scientific), citrate synthase (Abcam), and an oxidative phosphorylation (OXPHOS) cocktail (Abcam) overnight at 4 °C. Blots were then probed with horseradish peroxidase-conjugated anti-mouse (Jackson ImmunoResearch, West Grove, PA) or anti-rabbit (Thermo Fisher Scientific) secondary antibodies and detected using enhanced chemiluminescence (Bio-Rad).

Extraction of nuclear proteins and coimmunopreciptation were performed using the Universal Magnetic Co-IP Kit (Active Motif). Briefly, Aire-transfected HEK293 cells were lysed with hypotonic buffer and incubated on ice for 15 min. Cell lysates were centrifuged for 30 s at 14,000 x g, then the nuclei pellets were digested with an enzymatic shearing cocktail for 10 min at 37°C. After centrifugation of the nuclear lysates, the supernatants containing the nuclear proteins were first incubated with specific antibodies for 4 hr, then with Protein-G magnetic beads for 1 hr at 4°C with rotation. After four washes, bound proteins were eluted in laemmli/DTT buffer, separated by SDS/PAGE for 40 min at 200 V, transferred to PVDF membranes using the TurboTransfer System for 7 min at 25 V (BioRad) and blocked for 1 hr with TBS, 0.05% Tween, 3% milk. The western blot detection was done after incubation with primary (2 hr) and secondary antibodies (1 hr). Detection was performed by enhanced chemiluminiscence (ECL). Antibodies used for immunoprecipitation or revelation were: CLP1 (sc-243005, Santa Cruz), Flag-tag M2 (F1804, Sigma), goat IgG control (sc-2028, Santa Cruz), mouse IgG1 control (ab18443, Abcam), and horseradish peroxidase-conjugated anti-mouse IgG light chain specific (115-035-174, Jackson ImmunoResearch).

Cells lysates, prepared by dissolving washed cells directly in SDS sample buffer, were resolved by SDS-PAGE with indicated percentage of acrylamide. Western blotting was performed using standard protocols. For quantification, western blots were analyzed by ImageJ. Antibodies used in this paper as follows: Flag tag (M2; Sigma, St. Louis, MO), actin (Sigma), phospho CTD Tyr1 (3D12; Active Motif, Carlsbad, CA), U2AF65 (Sigma), histone H3 protein (abcam, Cambridge, MA), phospho CTD Ser2 (3E10; Millipore, Billerica, MA), phospho CTD Ser5 (3E8; Millipore), phospho CTD Ser 7 (4E12, Millipore), Rpb1 CTD (8WG16; abcam), GST tag (Invitrogen, Carlsbad, CA), Rpb1 (N20; Santa Cruz, Santa Cruz, CA), Exosc10 (Rrp6) (Novus, Littleton, CO), Exosc9 (Rrp45) (Novus), Exosc3 (Rrp40) (Novus), and Dis3 (Novus).