Ac 74

293T and HeLa cells were maintained in DMEM supplemented with 10% fetal calf serum (Gibco). An anti-p24 CA antibody has been described previously39 (link). Antibodies against HA (C29F4, Cell Signaling Technologies and HA.11, Covance), FLAG (M2, Sigma-Aldrich) and β-actin (AC-74, Sigma-Aldrich) were commercially available.

Immunoblotting was performed as described previously^{14 (link)}. In brief, cell lysates were generated from 2 × 10⁶ CTLs on day 2 of the cell culture. Protein samples were loaded on 10% gel for SDS-PAGE and were subsequently transferred to a PVDF membrane. Acetylated Histone H4 was detected using an anti-acetyl-histone H4 Ab (Millipore, 06-866). For detection of β-actin, a monoclonal anti-β-actin Ab (Sigma-Aldrich, AC-74) was used.

siRNA duplexes targeting COX7RP, OGDH, DLST, DLD, MDH1, and MDH2 were synthesized using an algorithm that significantly improves the target specificity of siRNA, in particular, by efficiently estimating off-target sequences^{48 (link)}. A non-targeting control siRNA (siControl) used in this study was purchased from RNAi Inc. (Tokyo, Japan)^{48 (link)}. The siRNA sequences were detailed in Supplementary Table 9. siRNAs were transfected into cells using RNAiMAX (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Cell lysates and mitochondrial fractions were prepared in a sample buffer for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), heated at 100 °C for 15 min, and subjected to SDS-PAGE and western blotting analysis using antibodies for COX7RP (1:1000 dilution)², RISP (1:5000 dilution, ab14746, Abcam, Cambridge, MA), COX1 (1:5000 dilution, ab14705, Abcam), COX4 (1:3000 dilution, 4844, Cell signaling, Danvers, MA), and β-actin (1:3000 dilution, AC-74, Sigma-Aldrich, St. Louis, MO).

RCC-PDC1 cells transfected with siRNAs or expression vectors were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, 1 mM PMSF, 1 μg/mL aprotinin) and the plasma membrane fraction was purified by centrifugation at 19,100g for 20 min at 4°C. Proteins were separated with SDS-PAGE and blotted on PVDF membrane, followed by reactions with anti-CXCR4 antibody (GTX22074, GeneTex) or anti-β-actin antibody (AC-74, SigmaAldrich) for loading control.

Western blot analysis was carried out using the Odyssey Infrared System (LI-COR Biosciences, Lincoln, NE) and anti-β-catenin antibody #9582 (Cell Signaling, Danvers, MA), anti-PIWIL1 antibody ab12337 (Abcam, Cambridge, MA), anti-HA antibody clone 16B12 (Covance, Berkeley, CA), monoclonal beta actin antibody AC-74 (Sigma, St. Louis, MO), and fluorescently-labeled secondary antibodies IRDye 680RD Infrared Dye (LI-COR Biosciences, Lincoln, NE). The experiments were repeated at least 3 times. Protein levels were normalized to actin levels (the loading control) with standard deviations.

Western blotting was performed as described previously [25] (link) with some modifications. Briefly, 8% to 16% gradient Tris-glycine SDS polyacrylamide gels were used (Thermo Fisher Scientific). Gel loading was normalized using antibodies AC-74 to β-actin or TUB 2.1 to β-tubulin (Sigma-Aldrich). After transfer to nitrocellulose membranes, blots were blocked in Blotto Blocker (Thermo Fisher Scientific) and incubated with primary antibodies, rabbit anti-p-EGFR (44–784 G, Thermo Fisher Scientific), mouse anti-p-p38 (ab50012, Abcam, Cambridge, MA), rabbit anti-p-ERK1/2 (4370, Cell Signaling) or rabbit anti-pAkt (9271, Cell Signaling). IRDye 800 CW or 680 RD goat anti-mouse or anti-rabbit (Li-Cor Biosciences, Lincoln, NE) was used as secondary antibodies. The blots were scanned using Odyssey CLX imaging system (Li-Cor Biosciences).