Mouse igg control antibody

Cells were collected, washed, and resuspended in MACS buffer (PBS with 5 mM EDTA, 2% (w/v) BSA). Then, cells were stained with an MCT1-antibody recognizing the N-terminus (Santa Cruz) at 1:200 in MACS buffer or a mouse IgG control antibody (Santa Cruz) for 1 h at room temperature. After extensive washing, cells were incubated with donkey antimouse Alexa Fluor^®-647 (Biolegend, Amsterdam, The Netherlands) at 1:500 dilution for 1 h at room temperature, followed by one wash step and fixation in 1% (v/v) formaldehyde in MACS buffer. Immunostaining was analyzed by fluorescence flow cytometry on a FACSVerseTM instrument (Becton Dickinson, East Rutherford, NJ, USA) using FACSuite Flow Cytometry Software (Becton Dickinson).

Preparation of DNA from SK-N-BE(2)C and Kelly cells for ChIP assay was performed using the Chromatin-Immunoprecipitation Assay kit (Merck Millipore) according to the manufacturer’s instructions. ChIP assays were performed with mouse anti-MYCN antibody (B84B; Santa Cruz Biotechnology); anti-ALYREF antibody (11G5; ImmunoQuest) or control mouse IgG antibody (Santa Cruz Biotechnology). DNA was purified using a MiniElute PCR Purification Kit (Qiagen). Real-time PCR was performed with primers designed to cover the regions of the ALYREF and USP3 genes containing MYC-responsive or ALYREF-responsive motifs or remote negative control regions. Fold enrichment of the ALYREF or USP3 genes containing the binding regions by the anti-MYCN or anti-ALYREF antibody was calculated by dividing the PCR product from this region by the PCR product from the negative control region, relative to the input.
For double-antibody ChIP assays, we performed sequential pull-downs, first with anti-MYCN antibody (B84B; Santa Cruz Biotechnology) followed by anti-ALYREF antibody (11G5, ImmunoQuest) using appropriate IgG controls.

For the detection of EGFR-Mfn1 complex, mitochondria fractions were obtained by Qproteome mitochondria isolation kit, and then subtracted in IP lysis buffer [20 mM Tris (pH 8.0), 150 mM NaCl, 100 μM Na₃VO₄, 50 mM NaF, 30 mM Na pyrophosphate, and 0.5% NP-40] containing cocktail of protease inhibitor (Roche Diagnostics, Basel, Switzerland). Mitochondrial lysates were incubated with EGFR IP-specific mouse monoclonal antibody (Cell Signaling, MA, USA) or control mouse IgG antibody (Santa Cruz Biotechnology, CA, USA) for 5 h at 4°C. Protein A-Sepharose beads (GE Healthcare, Little Chalfont, UK) were added to the immunoprecipitates and incubated at 4°C for 1 h. The beads were collected by centrifugation at 1,000 g for 3 min and washed 3 times in IP lysis buffer. Proteins were eluted with the SDS protein sample buffer before Western blotting with specific antibodies against Mfn1 and EGFR (Santa Cruz, CA, USA).

Cells were exposed to the proteasomal inhibitor MG132 (Sigma) at 20 μM concentration^{42 (link)}. Four hours post MG132 treatment, cells were lysed with denaturing lysis buffer (6 M guanidine-HCl, 50 mM sodium phosphate, pH 8.0, 500 mM NaCl, 5 mM imidazole). 5% of the protein was saved to serve as an input. The rest of the sample (750 μg total protein) was diluted in BC100 buffer (Sigma) freshly supplemented with protease inhibitor cocktail (Sigma) and subjected to immunoprecipitation with either 5 μg MYCN-specific antibody (Merck Millipore), or with 5 μg control mouse IgG antibody (Santa Cruz Biotechnology) and A/G PLUS agarose beads (Santa Cruz Biotechnology) at 4 °C overnight. Prior to immunoprecipitation, the beads were washed three times with denaturing wash buffer (8 M urea, 50 mM sodium phosphate, pH 8.0, 500 mM NaCl). After overnight incubation at 4 °C, beads were washed five times with cold BC100 buffer (Sigma) and once with cold sodium chloride solution (150 mM). Proteins were eluted in Laemmli sample buffer (62.5 mM Tris-HCl, pH 6.8; 25% glycerol; 2% SDS; 0.01% Bromophenol Blue) supplemented with β-mercaptoethanol (Bio-Rad) at a final concentration of 5%, and separated by electrophoresis using 10.5% or 10–14% Tris-HCl Criterion gels (Bio-Rad).

Preparation of DNA from SK-N-BE(2)-C and KELLY cells for ChIP assay were performed using the Chromatin-Immunoprecipitation Assay kit (Merck Millipore) according to the manufacturer’s instructions. ChIP assays were performed with mouse anti-MYCN antibody (B84B; Santa Cruz Biotechnology) or control mouse IgG antibody (Santa Cruz Biotechnology). DNA was purified using a MiniElute PCR Purification Kit (Qiagen). Real-time PCR was performed with primers designed targeting a negative control region (−2000 bp downstream of SNRPD3 TSS) or SNRPD3 target sequence (+647 bp upstream from SNRPD3 TSS). Fold enrichment of SNRPD3 target region was calculated by dividing cycle threshold values of the SNRPD3 target region by cycle threshold values of the negative control region, relative to input. Primer sequences (Supplementary Table 1) were obtained from Integrated DNA Technologies (NSW, Australia).