rabbit polyclonal anti pex14  (Proteintech)

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: Autophagy

Article Title: PEX13 prevents pexophagy by regulating ubiquitinated PEX5 and peroxisomal ROS

doi: 10.1080/15548627.2022.2160566

Figure Lengend Snippet: List of the antibodies used in the study.

Article Snippet: Anti-PEX14 (rabbit polyclonal) , 1:3000 , Millipore Sigma, ABC142.

Techniques: Concentration Assay, Western Blot, Immunofluorescence

Journal: Autophagy

Article Title: PEX13 prevents pexophagy by regulating ubiquitinated PEX5 and peroxisomal ROS

doi: 10.1080/15548627.2022.2160566

Figure Lengend Snippet: Depletion of PEX13 results in decreased peroxisome numbers. (A) HeLa cells were transfected with siRNA against PEX1, PEX2, PEX3, PEX5, PEX6, PEX10, PEX12, PEX13, PEX14, PEX19, PEX26 as well as a non-targeting control siRNA (siCTRL). Cells were fixed and immunostained for endogenous peroxisome marker ABCD3. Scale bar: 25 μm (B) Graph of ABCD3 density of images in (A). (n = 3; 30 cells/trial). Error bars represent standard deviation. (C) HeLa cells treated with siCTRL or si PEX13 , overexpression WT PEX13-MYC, W313G or I326T, stained anti-MYC and anti-ABCD3. Scale bar: 10 μm. (D) Quantification of images in (C) (n = 3; >30 cells/trial). Error bars represent standard deviation. Asterisks denote p-values compared to siCTRL: *p < 0.05, **p < 0.01.

Article Snippet: Anti-PEX14 (rabbit polyclonal) , 1:3000 , Millipore Sigma, ABC142.

Techniques: Transfection, Marker, Standard Deviation, Over Expression, Staining

Journal: Autophagy

Article Title: PEX13 prevents pexophagy by regulating ubiquitinated PEX5 and peroxisomal ROS

doi: 10.1080/15548627.2022.2160566

Figure Lengend Snippet: PEX13 depletion increases membrane localization and ubiquitination of PEX5. (A) HeLa cells transfected with siCTRL, si PEX1 , si PEX13 , or si PEX14 , fixed and stained for endogenous PEX5 (red) and ABCD3 (green). Prior to fixation cells were treated with 0.025% digitonin for 10 seconds to permeabilize the plasma membrane and washed with PBS. (B) Quantification of images in (A). Pearson’s correlation coefficient between PEX5 and ABCD3 was calculated (n = 3; 30 cells/trial). Error bars represent standard deviation. (C) Quantification of PEX5 on peroxisomes (A). Normalized mean fluorescent intensity of PEX5 within ABCD3 structures was calculated (n = 3; 30 cells/trial). Error bars represent standard deviation. (D) Flp-In T-Rex HEK293 cells stably expressing HA-tagged ubiquitin were transfected with siCTRL, si PEX13 or si PEX14 and treated with HCQ (10 μM) and doxycycline (1 μM) for 24 h. 3 h prior to lysis cells were treated with MG132 (10 μM). Immunoprecipitation (IP) with an antibody against the HA epitope tag was performed. Lysates and IP samples were separated by SDS-PAGE and probed for PEX5. (E) Immunofluorescent images of HeLa cells transfected with siCTRL or si PEX13 and co-transfected with USP30-Flag, fixed and stained for ABCD3 (red) and Flag (green). (F) Graph of ABCD3 density from images in (E) (n = 3; 30 cells/trial). Error bars represent standard deviation. (G) Immunoprecipitation of HA-ubiquitin in cells treated with the indicated siRNA as in D, however IP samples were treated with or without 5 mM BME and assessed on non-reducing SDS-PAGE. Full immunoblot of anti-PEX5 is presented in Figure S3B. Scale bars: 25 μm (A), 20 μm (F). Asterisks denote p-values; *p < 0.05, **p < 0.01.

Article Snippet: Anti-PEX14 (rabbit polyclonal) , 1:3000 , Millipore Sigma, ABC142.

Techniques: Transfection, Staining, Standard Deviation, Stable Transfection, Expressing, Lysis, Immunoprecipitation, SDS Page, Western Blot

Journal: Autophagy

Article Title: PEX13 prevents pexophagy by regulating ubiquitinated PEX5 and peroxisomal ROS

doi: 10.1080/15548627.2022.2160566

Figure Lengend Snippet: PEX1 and PEX13 depletion induces autophagy, decreases CAT localization to peroxisomes and increases cellular ROS. (A) HeLa cells over-expressing GFP-MAP1LC3B, transfected with siCTRL, si PEX1 , si PEX13 , si PEX14 alone, or co-transfected with si NBR1 . (B) Quantification of images in (A). The number of cells with more than 10 GFP-MAP1LC3B puncta were compared between the siRNA treatments (n = 3; 30 cells/trial). Error bars representing standard deviation. (C) Immunofluorescent images of HeLa cells treated with the indicated siRNAs stained for endogenous catalase (red) and ABCD3 (green). (D) Quantification of mean catalase intensity within ABCD3 structures of images in (A) (n = 3; 30 cells/trial). Error bars represent standard deviation. (E) Membrane fractionation of HEK293 cells treated with indicated siRNA, probed for catalase and ABCD3. Cyto denotes cytosolic fraction, Meb denotes membrane fraction. (F) Live cell images of HeLa cells treated with indicated siRNA and ROS probe OxyBURST. (G) Quantification of mean fluorescent intensity of images in (F) (n = 3; 30 cells/trial). Error bars representing standard deviation. Asterisks denote p-values; *p < 0.05, **p < 0.01. Scale bars: 10 μm (A), 25 μm (C), 50 μm (F).

Article Snippet: Anti-PEX14 (rabbit polyclonal) , 1:3000 , Millipore Sigma, ABC142.

Techniques: Expressing, Transfection, Standard Deviation, Staining, Fractionation

Journal: Autophagy

Article Title: PEX13 prevents pexophagy by regulating ubiquitinated PEX5 and peroxisomal ROS

doi: 10.1080/15548627.2022.2160566

Figure Lengend Snippet: List of the forward sequences of siRNA used in the study.

Article Snippet: Anti-PEX14 (rabbit polyclonal) , 1:3000 , Millipore Sigma, ABC142.

Techniques: Sequencing

Journal: Autophagy

Article Title: PEX13 prevents pexophagy by regulating ubiquitinated PEX5 and peroxisomal ROS

doi: 10.1080/15548627.2022.2160566

Figure Lengend Snippet: List of the antibodies used in the study.

Article Snippet: Anti-PEX14 (rabbit polyclonal) , 1:3000 , Millipore Sigma, ABC142.

Techniques: Concentration Assay, Western Blot, Immunofluorescence