polyclonal rabbit antibodies against pex14  (Merck KGaA)

Journal: Genetics in medicine : official journal of the American College of Medical Genetics

Article Title: Autosomal dominant Zellweger spectrum disorder caused by de novo variants in PEX14 gene.

doi: 10.1016/j.gim.2023.100944

Figure Lengend Snippet: Figure 1 Analysis of patients’ fibroblasts. A. Immunofluorescence microscopy analysis to determine the subcellular location of the peroxisomal matrix protein catalase (green signal) and the peroxisomal membrane protein ABCD3 (red signal) in primary fibroblasts of the 2 heterozygous PEX14 patients (patient 1 and 2), 2 previously reported PEX14-null patients (PEX14(1) and PEX14(2)), and a control indi- vidual. Shown are representative images (scale bar, 10 μm). B. Electropherograms showing the Sanger sequencing results of patient 1 and patient 2 and their respective parents. Both patients are heterozygous for a de novo PEX14 variant (location indicated by arrows). Patient 1 is heterozygous for NC_000001.11(NM_004565.2):c.585+1G>T and patient 2 is heterozygous for NM_004565.2:c.585G>A. C. Schematic

Article Snippet: Immunoblot analysis was performed with rabbit polyclonal antibodies against PEX14 (gift from M. Fransen; diluted 1:1,000), peroxisomal 3-ketoacyl-CoA thiolase (Atlas antibodies HPA007244; diluted 1:2,000), ACOXI (Abcam ab184032; diluted 1:2,000), ABCD1 (Euromedex; diluted 1:500), ABCD3 (Thermo Fisher PA1-650; diluted 1:2,000), ACBD5 (Sigma-Aldrich HPA012145; diluted 1:500), PEX5 (Sigma HPA039259; diluted 1:500), PEX13 (gift from D. Crane; diluted 1:1,000), PEX19 (Sigma-Aldrich; diluted 1:1,000), LC3B (Abcam ab48394; diluted 1:1,500), or SQSTM1/P62 (BD Transduction lab; diluted 1:2,000).

Techniques: Microscopy, Membrane, Control, Sequencing, Variant Assay

Journal: Genetics in medicine : official journal of the American College of Medical Genetics

Article Title: Autosomal dominant Zellweger spectrum disorder caused by de novo variants in PEX14 gene.

doi: 10.1016/j.gim.2023.100944

Figure Lengend Snippet: Figure 2 Analysis of truncated PEX14 protein. A. Immunofluorescence microscopy analysis of control HeLa cells transfected with a cDNA encoding PEX14-trunc results in a peroxisomal mosaic phenotype similar as observed in the patient cells (compare with Figure 1A) (scale bar, 10 μm). Transfection of HeLa cells with a cDNA encoding PEX14-wt does not result in a peroxisomal mosaic phenotype (not shown). B. Pull- down experiments with HEK 293 cells expressing FLAG-tagged PEX14-wt or FLAG-tagged PEX14-trunc. As a control, we included HEK 293 cells transfected with the expression vector lacking PEX14 (empty vector). FLAG-tagged PEX14 proteins and associated proteins were immunoprecipitated using FLAG-specific antibodies followed by immunoblot analysis of the immunoprecipitates using antibodies against PEX5, PEX13, PEX14, and PEX19. Performed in triplicate; shown are representative immunoblots. C. Immunofluorescence microscopy analysis to determine the subcellular location of the peroxisomal matrix protein import receptor PEX5 (green signal) in primary fibroblasts of the heterozygous PEX14 patients, a PEX14-null patient (PEX14(2)), a PEX13-null patient, and a control individual. Peroxisomal membranes are stained with antibodies against the peroxisomal membrane protein ABCD3 (red signal). Shown are representative images (scale bar, 10 μm).

Article Snippet: Immunoblot analysis was performed with rabbit polyclonal antibodies against PEX14 (gift from M. Fransen; diluted 1:1,000), peroxisomal 3-ketoacyl-CoA thiolase (Atlas antibodies HPA007244; diluted 1:2,000), ACOXI (Abcam ab184032; diluted 1:2,000), ABCD1 (Euromedex; diluted 1:500), ABCD3 (Thermo Fisher PA1-650; diluted 1:2,000), ACBD5 (Sigma-Aldrich HPA012145; diluted 1:500), PEX5 (Sigma HPA039259; diluted 1:500), PEX13 (gift from D. Crane; diluted 1:1,000), PEX19 (Sigma-Aldrich; diluted 1:1,000), LC3B (Abcam ab48394; diluted 1:1,500), or SQSTM1/P62 (BD Transduction lab; diluted 1:2,000).

Techniques: Microscopy, Control, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, Staining, Membrane

Journal: Genetics in medicine : official journal of the American College of Medical Genetics

Article Title: Autosomal dominant Zellweger spectrum disorder caused by de novo variants in PEX14 gene.

doi: 10.1016/j.gim.2023.100944

Figure Lengend Snippet: Figure 5 Rescue of functional peroxisomes by late autophagy inhibitor chloroquine and genetic knockdown of NBR1. A. Immu- noblot analysis of homogenates prepared from primary fibroblasts of the 2 heterozygous PEX14 patients (patient 1 (P1) and patient 2 (P2)), 1 previously reported PEX14-null patient (PEX14(2)), and 2 control individuals (C1 and C2) cultured at 37 ◦C and 40 ◦C. Cells were incubated with 10 μM chloroquine (CQ) for 7 days. Immunoblots were stained with antibodies against the peroxisomal matrix protein thiolase, the peroxisomal membrane protein ABCD1, the autophagy adaptor protein SQSTM1/P62 (P62), MAPILC3B (LC3I and LC3II), and, as loading control, tubulin. Performed in triplicate; shown are representative immunoblots. B. Percentage of patients’ cells with catalase- or ABCD3- stained peroxisomes when cultured at 37 ◦C (white bars), or 7 days at 40 ◦C in the absence (dashed bars) or presence (stippled bars) of 10 mM CQ. Cells were examined with immunofluorescence microscopy to determine the subcellular location of the peroxisomal matrix protein catalase and the peroxisomal membrane protein ABCD3 (for examples, see Supplemental Figure 2). Performed in quadruplicate; for each condition 100 cells were examined (total of 400 cells) (Two-way anova with Tukey’s multiple comparisons test, P = .0332, indicated by *,

Article Snippet: Immunoblot analysis was performed with rabbit polyclonal antibodies against PEX14 (gift from M. Fransen; diluted 1:1,000), peroxisomal 3-ketoacyl-CoA thiolase (Atlas antibodies HPA007244; diluted 1:2,000), ACOXI (Abcam ab184032; diluted 1:2,000), ABCD1 (Euromedex; diluted 1:500), ABCD3 (Thermo Fisher PA1-650; diluted 1:2,000), ACBD5 (Sigma-Aldrich HPA012145; diluted 1:500), PEX5 (Sigma HPA039259; diluted 1:500), PEX13 (gift from D. Crane; diluted 1:1,000), PEX19 (Sigma-Aldrich; diluted 1:1,000), LC3B (Abcam ab48394; diluted 1:1,500), or SQSTM1/P62 (BD Transduction lab; diluted 1:2,000).

Techniques: Functional Assay, Knockdown, Control, Cell Culture, Incubation, Western Blot, Staining, Membrane, Microscopy

Journal:

Article Title: Identification of a Novel, Intraperoxisomal Pex14-Binding Site in Pex13: Association of Pex13 with the Docking Complex Is Essential for Peroxisomal Matrix Protein Import

doi: 10.1128/MCB.25.8.3007-3018.2005

Figure Lengend Snippet: Dependence of peroxisomal matrix protein import on direct Pex13-Pex14 interactions. Oleic acid-induced wild-type and pex13Δ cells expressing HA-Pex11 were subjected to double-immunofluorescence microscopy so as to localize HA-Pex11 in tandem with PTS1 protein Pcs60 (A), PTS2 protein Fox3 (B), or PTS1-like protein Cta1 (C). The same procedure was applied to pex13Δ cells expressing Pex13 (pKat113), Pex13SH3 (pAS53), Pex13loop (pAS71), and Pex13loop+SH3 (pAS73). Detection was achieved with mouse monoclonal antibodies against the HA epitope combined with rabbit polyclonal antibodies against the individual matrix proteins. As secondary antibodies, Alexa Fluor 488-labeled anti-mouse IgG and Alexa Fluor 594-labeled anti-rabbit IgG were used. A congruent fluorescence pattern denotes colocalization of HA-Pex11 with the analyzed matrix proteins, a finding which is readily revealed in the merged images. (D) From the same cells, a postnuclear supernatant (PNS) was produced and subfractionated into a 25,000 × g pellet fraction (P) enriched for peroxisomes and a supernatant fraction (SN) enriched for the cytosol. Equivalent volumes of fractions were loaded on the gels, transferred to nitrocellulose membranes, and analyzed for the distributions of the specified proteins.

Article Snippet: Colocalization studies carried out with oleic acid-induced yeast cells were performed with an Axioplan microscope and AxioVision 4.1 software (Zeiss, Jena, Germany) as described previously ( 19 ) and with the previously described rabbit polyclonal antibodies against Pex14, Fox3 ( 15 ), Pcs60 ( 4 ), and Cta1 ( 25 ) as well as mouse monoclonal antibodies against the hemagglutinin (HA) epitope (Santa Cruz Biotechnology, Santa Cruz, Calif.).

Techniques: Expressing, Immunofluorescence, Microscopy, Labeling, Fluorescence, Produced

Journal:

Article Title: Identification of a Novel, Intraperoxisomal Pex14-Binding Site in Pex13: Association of Pex13 with the Docking Complex Is Essential for Peroxisomal Matrix Protein Import

doi: 10.1128/MCB.25.8.3007-3018.2005

Figure Lengend Snippet: Dependence of matrix protein import on Pex5 in the absence of direct Pex13-Pex14 interactions. (A) Oleic acid-induced pex5Δ pex13Δ cells expressing mutant versions of Pex13 in combination with HA-Pex11 were processed for double-immunofluorescence microscopy with rabbit antibodies against PTS2 protein Fox3 and anti-HA monoclonal antibodies. The secondary antibodies used were Alexa Fluor 488-labeled anti-mouse IgG and Alexa Fluor 594-labeled anti-rabbit IgG. pex5Δ cells were used as a control for intact PTS2 import. (B and C) The same cells but additionally expressing Pex5W204A were similarly inspected for the localization of Fox3 (B) and Pcs60 (C).

Article Snippet: Colocalization studies carried out with oleic acid-induced yeast cells were performed with an Axioplan microscope and AxioVision 4.1 software (Zeiss, Jena, Germany) as described previously ( 19 ) and with the previously described rabbit polyclonal antibodies against Pex14, Fox3 ( 15 ), Pcs60 ( 4 ), and Cta1 ( 25 ) as well as mouse monoclonal antibodies against the hemagglutinin (HA) epitope (Santa Cruz Biotechnology, Santa Cruz, Calif.).

Techniques: Expressing, Mutagenesis, Immunofluorescence, Microscopy, Labeling