Pmp70

Adult male mice, from both genotypes, were killed by an overdose of isoflurane at the same time (ZT3) to avoid possible circadian variation of protein expression. Brains were removed quickly and snap frozen. Protein was extracted by homogenizing the hippocampus in lysis buffer (T-PER Protein Extraction Reagent, 1% Halt Protease Inhibitor Cocktail (Thermo scientific, USA)) using electric homogenizer. Homogenates were centrifuged at 16000 g at 4°C for 20 minutes, and then the supernatant was collected. Protein concentration was determined using Bradford protein assay [68 (link)]. Proteins were separated on 8% SDS-PAGE, and then transferred to Invitrolon^TM PVDF membrane (Life technologies, CA). Membrane was blocked using StartingBlock^TM (TBS) blocking buffer (Thermo scientific, USA), then incubated with rabbit polyclonal anti PMP70 (1:1000, SIGMA-ALDRICH, USA) or rabbit polyclonal anti actin (1:1000, SIGMA-ALDRICH, USA) overnight at 4°C. This was followed by washing then incubation with HRP-conjugated goat anti rabbit IgG one hour at room temperature. Immunopositive bands were detected using Immobilon^TM Western (Millipore, USA) and visualized by ChemiDoc^™ MP System (Biorad).

Mouse monoclonal antibodies against the peroxisomal membrane protein PMP70 (Sigma, St. Louis, MO), HIV-1 p24 (Abcam, Cambridge, MA), GFP (Abcam), myc (Millipore Sigma, Etobicoke, ON, Canada), and beta-actin (Abcam) were purchased from the indicated suppliers. Rabbit polyclonal antibodies to PEX7, PEX11B, PEX13, PEX19, catalase, beta-catenin, and AU1 were obtained from Abcam; rabbit polyclonal antibody to PEX2 (PXMP3) was purchased from Pierce (Rockford, IL); rabbit monoclonal antibody to β-TrCP (D12C8) was purchased from New England Biolabs (Whitby, ON, Canada); and rabbit polyclonal antibody to the tripeptide SKL was produced as previously described (61 (link)).
Donkey anti-mouse IgG conjugated to Alexa Fluor 680, goat anti-rabbit IgG conjugated to Alexa Fluor 800, donkey anti-mouse IgG conjugated to Alexa Fluor 488, donkey anti-rabbit IgG conjugated to Alexa Fluor 488, donkey anti-rabbit IgG conjugated to Alexa Fluor 546, and donkey anti-mouse IgG conjugated to Alexa Fluor 546 were purchased from Invitrogen.

For the immunofluorescence experiments, the following antibodies were used: MCT2 (sc-14926; Santa Cruz Biotechnology, Santa Cruz, CA, USA), MCT1 (sc-365501; Santa Cruz Biotechnology), MCT4 (sc-50329; Santa Cruz Biotechnology), CD147 (sc-71038; Santa Cruz Biotechnology), gp70 (HPA017740, 1:100; Atlas Antibodies, Stockholm, Sweden), PEX14 (a gift from Dr. Dennis Crane, Griffith University, Brisbane, Australia), catalase (ab88650; Abcam, Cambridge, UK), TRITC (Jackson ImmunoResearch, West Grove, PA, USA) and Alexa 488 (Invitrogen, Life Technologies, Carlsbad, CA, USA). For the Western blot analysis, the following antibodies were used: MCT2, ACOX1 (a gift from A. Völkl, University of Heidelberg, Germany), ACOX3 (sc-135435; Santa Cruz Biotechnology), PEX14, PMP70 (SAB4200181; Sigma-Aldrich, St Louis, MO, USA) and α-Tubulin (T9026, Sigma-Aldrich). For the immunohistochemistry (IHC) staining, the following antibodies were used: AMACR (504R-16, Cell Marque, Rocklin, CA, USA), ACOX3 (sc-135435; Santa Cruz Biotechnology) and DBP (a gift from Dr. Gabriele Moller from HelmholtzZentrum München). Pex19-YFP plasmid was a gift from Dr M. Schrader, Exeter University, UK.