Normal mouse immunoglobulin g igg

Paraquat (MP Biomedicals, Santa Ana, CA), A23187, and ionomycin (Calbiochem/Merck, Darmstadt, Germany) were used at the indicated concentrations. Phorbol myristic acid (Calbiochem) was used at 1 μM. CCCP, antimycin A, valinomycin, and bafilomycin A1 were from Sigma-Aldrich (St. Louis, MO) and used at final concentrations of 20 μM, 40 μg/ml, 10 μM, and 100 nM, respectively. Staurosporine (Tocris Biosciences, Bristol, UK) was used at 2 μM. Mff and Mfn1 antibodies were described previously (Gandre-Babbe and van der Bliek, 2008 (link); Tanaka et al., 2010 (link)). The following antibodies were purchased: FLAG (Genescript, Piscataway, NJ), HA (Roche, Basel, Switzerland), Tom20, Tom40, Parkin, and normal mouse immunoglobulin G (IgG; Santa Cruz Biotechnology, Santa Cruz, CA), calnexin and Drp1 (BD Transduction Laboratories, Franklin Lakes, NJ), p62 (Abnova, Taipei City, Taiwan), porin (MitoSciences, Eugene, OR), LC3B (Sigma-Aldrich), Fis1 (Alexis Biochemicals and Proteintech, Farmingdale, NY), α-tubulin (Invitrogen, Carlsbad, CA), and actin (Sigma-Aldrich). Rabbit polyclonal antibodies were generated against polypeptide fragments of C. elegans DRP-1 protein and used along with other antibodies to probe blots of C. elegans extracts as described previously (Head et al., 2011 (link)).

Cells were lysed and the nuclear fraction was isolated using Subcellular Protein Fractionation Kit (Thermo Fisher Scientific, no. 78840). Nuclear extracts were incubated with two independent HOXD11 antibodies (Santa Cruz Biotechnology, no. sc-81969 and Sigma-Aldrich, no. SAB1403944) at 4°C overnight with rotation. Normal mouse immunoglobulin G (IgG) (Santa Cruz Biotechnology, no. sc-2025) was used as a negative control. Fifty microliters of protein A agarose bead slurry (Invitrogen) were added to the IP complexes and incubated at 4°C for 2 hours. The beads were washed twice with in lysis buffer, pelleted, eluted in 2× SDS Laemmli buffer, and separated by electrophoresis. Protein levels of PBX1 were assessed by immunoblotting in HOXD11-IP complexes, protein lysates, and nuclear fractions of ELT3 cells.