Journal: bioRxiv

Article Title: Loss of PREPL alters lipid homeostasis due to mitochondrial defects

doi: 10.1101/2025.10.28.685080

Figure Lengend Snippet: (A) Co-staining of PREPL (red) with the peroxisomal marker PEX14 (green). The nuclei were counterstained with DAPI (blue) (B) Peroxisome staining in WT and PREPL KO HEK293T cells (red: PEX14; blue: DAPI). (C) Quantification of peroxisome number per cell and peroxisome length in WT and PREPL KO HEK293T cells. (D) quantitative western blot of peroxisomal proteins in WT and PREPL KO HEK293T cells. Statistical analysis was performed using a T-test * p < 0.05, ** p < 0.01.

Article Snippet: The antibodies used were directed against GAPDH (Cell Signaling Technology, 2118), PEX14 , catalase (Calbiochem, 219010), ACAA1 (Atlas Antibodies, HPA007244), and HSD17B4 (Fisher Scientific, 15116-1-AP).

Techniques: Staining, Marker, Western Blot

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: The FEBS journal

Article Title: Membrane topologies of PEX13 and PEX14 provide new insights on the mechanism of protein import into peroxisomes.

doi: 10.1111/febs.14697

Figure Lengend Snippet: Figure 1 – Schematic representation of PEX13 and PEX14 and their interactions with each other and with PEX5. The amino acid positions of the globular domains of Rattus Norvegicus PEX13 and PEX14 proteins are indicated (CC, Coiled coil; NTD, N-terminal domain of PEX14; SH3, Src Homology 3 domain). Putative transmembrane domains (TMDs; light blue cylinders) were predicted with PRALINE™ [83] and PHOBIUS [84] using an alignment of twenty protein sequences representative of eukaryotic evolution. The pentapeptide motifs of PEX5 [43] are represented in orange. Black lines indicate the epitopes recognized by some of the antibodies used in this work. The interactions between PEX13, PEX14 and PEX5 are indicated as red (yeast) or blue (mammals) arrows. The SH3 domain of yeast PEX13 interacts with a PXXP motif (residues 87-90) located between the NTD and TMD of PEX14 [61,85]. In mammals, the SH3 domain of PEX13 interacts with the NTD of PEX14 [41,63]. The region comprising amino acid residues 236-246 of yeast PEX13, which corresponds to a region in the mammalian peroxin located between TMD2 and TMD3, interacts with an undefined region (marked with “?”) of PEX14 [47]. The SH3 of PEX13 also interacts with the N-terminal half of PEX5 in yeast [36,37,85]. A similar interaction involving residues 219-403 of human PEX13 (which includes the SH3 domain) and the N-terminal half of PEX5 has been described [38]. Residues 1-135 of mammalian PEX13 were also reported to interact with pentapeptides motifs 2-4 of PEX5 [45]. The NTD of PEX14 interacts with pentapeptide motifs present in the N-terminal half of PEX5 both in mammals and yeast/fungi [43,45,49,71,86]. A C- terminal domain of yeast PEX14 (residues 235-341) has been shown to interact with PEX5 [49,71], an interaction involving the second pentapeptide motif of PEX5 [49] (Reviewed in [51,57,70]).

Article Snippet: The following antibodies were used: rabbit antibody against full-length human PEX14 protein [14], rabbit antibody against amino acid residues 327-377 of human PEX14 protein (Bethyl laboratories.

Techniques:

Journal: The FEBS journal

Article Title: Membrane topologies of PEX13 and PEX14 provide new insights on the mechanism of protein import into peroxisomes.

doi: 10.1111/febs.14697

Figure Lengend Snippet: Figure 2 – PEX14 is a transmembrane protein with a Nin-Cout topology. A- Expression and purification of H6TEVPEX14 using IMAC. Culture aliquots collected before (lane NI) and after (lane I) protein induction, lysed cell extract (lane T) and the corresponding soluble (lane S) and insoluble protein fractions (lane P), the non-bound protein fraction from the Nickel-NTA beads (NB) and the purified protein () – H6TEVPEX14 (lane P14) were analyzed by SDS-PAGE, blotted onto a nitrocellulose membrane, and stained with Ponceau S. B- Proteoliposomes (Lipos) containing either PEX14 (left panel) or H6TEVPEX14 (H6PEX14; right panel) were treated or not with PK (400 µg/mL) in the presence or absence of TX-100, and analyzed by SDS-PAGE/Western blot. PEX14 was detected with antibodies against the full-length (FL) protein. Equivalent amounts of recombinant proteins (Lanes I; 130 ng) and the corresponding reconstituted proteins were loaded onto the gels. C- As in B, but using PEX14 that was subjected to the reconstitution protocol in the absence of lipids. D- As in B, but using antibodies directed to the full-length protein, the C-terminal residues 327-377 of human PEX14, and the N-terminal histidine-tag. E- PEX14 proteoliposomes were treated with PK (400 µg/mL) or trypsin (Try, 400 µg/mL) and analyzed by SDS-PAGE/Western blot using an antibody against the full-length protein. The ~46-kDa PEX14 fragment obtained after trypsin digestion is recognized by the anti-PEX14(327-377) antibody (data not shown). Identical results were obtained with H6TEVPEX14; in this case, the two fragments with ~20 and ~30 kDa are recognized by the anti-His antibody (data not shown), suggesting that the 30-kDa fragment is a partial proteolysis product. F- Proteoliposomes, PK-treated proteoliposomes, and PK alone were subjected to SDS- PAGE, blotted onto a Sequi-blot PVDF membrane and stained with Coomassie Brilliant Blue. PK- resistant fragments of PEX14 (, 40 kDa and ~20 kDa) and protein bands derived from PK itself (*)

Article Snippet: The following antibodies were used: rabbit antibody against full-length human PEX14 protein [14], rabbit antibody against amino acid residues 327-377 of human PEX14 protein (Bethyl laboratories.

Techniques: Expressing, Purification, SDS Page, Membrane, Staining, Western Blot, Recombinant, Derivative Assay