rabbit polyclonal antibody to pex14  (Novus Biologicals)

Journal: npj Viruses

Article Title: The Wnt/β-catenin pathway is important for replication of SARS-CoV-2 and other pathogenic RNA viruses

doi: 10.1038/s44298-024-00018-4

Figure Lengend Snippet: A A549 cells were treated with DMSO alone or ten different commercially available drugs/compounds (1 μM) that block Wnt/β-catenin signaling. Cells were fixed at 24- and 48-h post-drug treatment and processed for confocal microscopy using an antibody against PEX14 to label peroxisomes and CellMask TM to label the plasma membrane. The numbers of peroxisomes in cells were determined using Volocity software. The peroxisome density (number/μm 3 ) was calculated by dividing the number of peroxisomes by the estimated cell volume. For each sample, peroxisome densities were determined using a minimum of 20 cells. Data from three independent experiments are shown. Error bars represent standard errors of the mean. Two-way ANOVA with Bonferroni post-hoc tests were used to determine significance between samples treated with DMSO and Wnt inhibitors. * P < 0.05; ** P < 0.01. B Calu-3 cells were treated with the indicated Wnt inhibitors (1 μM) or Pyrvinium (100 nM) for 48-h and then total RNA, including small RNAs, were extracted from the samples, and relative levels of miRNAs were determined by RT-qPCR. The average relative levels of miRNAs (normalized to snRNU6) from three independent experiments were determined. Two-way ANOVA with Bonferroni post-hoc tests was used to determine significance between samples treated with DMSO and Wnt inhibitors. Error bars represent standard errors of the mean. *** P < 0.001.

Article Snippet: Primary antibodies were from the following sources: mouse monoclonal against beta-actin (ab8224), rabbit monoclonal against PEX7 (ab133754), rabbit polyclonal antibodies to PEX2 (ab110004), PEX13 (ab190213), PEX11B (ab211508), PEX19 (ab137072), β-catenin (ab32572), and catalase (ab1877) from Abcam (Cambridge, MA); Mouse monoclonal antibody to SARS-CoV/SARS-CoV-2 (COVID-19) spike antibody 1A9 (GTX632604) and rabbit polyclonal antibody to SARS-CoV-2 nucleocapsid (GTX135357) from GeneTex (Irvine, CA); Rabbit polyclonal antibody to PEX14 (NBP2-33455) from Novus Biologicals (Centennial, CO, USA); Mouse monoclonal antibody against the peroxisomal membrane protein PMP70 (SAB4200181) from Sigma (St. Louis, MO).

Techniques: Blocking Assay, Confocal Microscopy, Clinical Proteomics, Membrane, Software, Quantitative RT-PCR

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