Rpn2106

QhTERT cells that overexpress either FGFR2b or FGFR2c were seeded in 12-well flat-bottom plates with 500 μL of serum-free medium for 16 hours. FGF1 (#2232-FA-025, R&D systems) was reconstituted to a concentration of 100 μg/mL using PBS, diluted with 0.1% bovine serum albumin, and added to the cells at final concentrations of 100 ng/mL for 20 min in separate wells. Heparin (# H3149-10KU, Sigma) with final concentration of 100 units/mL was also added to increase stability. In addition, peptides at concentrations of 5 and 100 μM were incubated for 20 min in separate wells. The cells were washed with PBS and lysed in RIPA buffer containing protease inhibitors (#11836170001, Roche, Basel, Switzerland). Lysates were separated by gel electrophoresis, transferred to polyvinylidene difluoride membranes (#ISEQ00010, Millipore), and detected by immunoblotting using an enhanced chemiluminescence system (#RPN2106, GE Healthcare). Anti-FGFR2 antibody (#SC122, Santa Cruz Biotechnology), anti-phospho-FGFR (#3471, Cell Signaling Technology), anti-AKT (#4691P, Cell Signaling Technology), anti-ERK1/2 (#4695P, Cell Signaling Technology), anti-phospho-AKT (pS473; #4060P, Cell Signaling Technology), anti-phospho-ERK1/2 (#4370P, Cell Signaling Technology), and anti-tubulin (#32–2600, Invitrogen) were used as per manufacturer's instructions.

For analyzing the DNA binding affinity of each CP2c-containing complex, nuclear lysates from transfected 293T cells were mixed with biotin-conjugated double-stranded DNA probes in a concentration dependent manner (0~100 nM). Samples with various combinations were incubated for 2 h at room temperature with Streptavidin-Sepharose beads (Invitrogen, Waltham, MA, USA, 15942-050). Pull down samples were washed with lysis buffer once. For immunoblotting, protein loading samples were prepared by boiling in 2Χ SDS-PAGE sample loading buffer. Proteins were visualized by chemiluminescence using an ECL system (GE healthcare, RPN2106). Relative amounts of proteins were quantified using the Image J (ver. 1.51) program. The proportion of protein bound to DNA was derived by calculating the band intensity of Western blot as output versus input. Relative amounts of CP2c in the tCP2c were obtained by subtracting the amounts of CP2b or Pias1 from the total amounts of CP2c, assuming that the amounts of CP2c in the CBP were the same as those of CP2b or Pias1 [23 (link)].

Whole muscle samples (100 ng proteins) were subjected to 12.5% SDS-PAGE under reducing conditions and transferred onto Amersham Hybond ECL nitrocellulose membranes (RPN2020D; GE Healthcare, Little Chalfont, UK), as described previously [22 (link)]. The blots were blocked with 10% powdered milk in 0.1% (v/v) polyethylene sorbitan monolaurate (Tween 20)-Tris buffered saline (TTBS) before incubation with a mouse monoclonal primary antibody cocktail of anti-myoglobin (clone MG-1; M7773, Sigma, St. Louis, MO, USA; 1:1000 dilution) [23 (link), 24 (link)] and anti-α-tubulin (clone DM1A; T9026, Sigma; 1:1000) [25 (link), 26 (link)] in CanGetSignal solution 1 (NKB-101; Toyobo, Osaka, Japan) overnight at 4°C. Membranes were treated for 1 h with biotinylated anti-mouse IgG secondary antibody (BA-9200; Vector Labs., Burlingame, CA, USA) at 1:5000 dilution in CanGetSignal solution 2 (NKB-101; Toyobo) and with horseradish peroxidase (HRPO)-labeled avidin (PK-6100; Vector Labs.) at 1:500 dilution in TTBS for 30 min at room temperature, followed by enhanced chemiluminescence (ECL) detection (RPN2106; GE Healthcare) onto Amersham Hyperfilm ECL (28906837; GE Healthcare) according to the manufacturer’s recommendation. Myoglobin band intensity was quantified by densitometry normalized to α-tubulin as an internal standard.

Proteins were separated by SDS–PAGE under reducing conditions and transferred to poly(vinylidene fluoride) membranes. Membranes were blocked with 5% nonfat milk in phosphate-buffered saline with 0.1% Tween 20 (Sigma-Aldrich, P9416; PBST). The blots were incubated overnight at 4 °C with primary antibodies. Immunoblot analysis was performed using an enhanced chemiluminescence procedure (GE Healthcare, RPN2106), and western blot images were captured using the ChemiDoc™MP system (Bio-Rad), Image Lab version 6.1.0. Densitometric analysis was performed using ImageJ software (National Institutes of Health). The following antibodies were used: Cell Signaling Technology: anti-COXIV (3E11, #4850), 1:1000; anti-VDAC1 (D73D12, #4661), 1:1000; anti-Tomm20 (D8T4N, #42406), 1:1000; anti-GAPDH (14C10, #2118), 1:10000; anti-Cytochrome c (136F3, #4280), 1:1000; anti-LC3B (#2775), 1:1000; Novus Biologicals: anti-Dhps (NBP1-82648), 1:1000; BD Biosciences: anti-Eif5a (611976), 1:1000; Sigma-Aldrich: anti-Dohh (HPA041953), 1:1000; Millipore: anti-Hypusine (ABS1064), 1:1000; anti-puromycin (12D10, #MABE343), 1:1000; Abcam: anti-Tfam (ab131607), 1:1000; anti-PGC1α (ab54481), 1:1000; anti-TFEB (ab2636), 1:1000; and Atp5a, Uqcrc2, Sdhb, and Ndufb8 were detected using Total OXPHOS Rodent WB Antibody Cocktail (ab110413), 1:1000. Primary antibodies were diluted in 1% BSA containing PBST.