plasmid ha-p62  (Addgene inc)

Journal: Virology journal

Article Title: Usutu virus NS4A induces autophagy and is targeted by the selective autophagy receptor p62/SQSTM1 for degradation.

doi: 10.1186/s12985-025-02719-5

Figure Lengend Snippet: Fig. 5 p62 has an antiviral effect during USUV replication and modulates the ISG response. A549 cells were transfected with siRNA targeting p62 or the non-targeting pool (NTP). At 48 hpt cells were infected with USUV (MOI = 1). At 24 hpi supernatants and lysates were collected. A Knockdown of p62 was confirmed by western blot analysis. B-C Viral mRNA copies (B) and infectious progeny (C) were determined by RT-qPCR and plaque assay, respectively. Means ± standard deviation of 5 independent experiments are shown and statistical significance relative to NTP-treated cells is indicated (** p < 0.01). D-F Total RNA was isolated from the lysates and IFN-β (D), IFIT2 (E), and ISG15 (F) mRNA levels were measured by RT-qPCR. Gene expression was normalized to RPL13a and the fold change versus NTP-infected cells calculated. Means ± standard deviation of 3–4 independent experiments are shown and statistical significance relative to NTP-treated cells is indicated (* p < 0.05, **** p < 0.0001)

Article Snippet: The p62-HA plasmid [30] was obtained from Addgene (#28027) and it was modified by replacing the HA tag with a C-terminal FLAG tag.

Techniques: Transfection, Infection, Knockdown, Western Blot, Quantitative RT-PCR, Plaque Assay, Standard Deviation, Isolation, Gene Expression

Journal: bioRxiv

Article Title: Molecular mechanisms underlying p62-dependent secretion of the Alzheimer-associated ubiquitin variant, UBB +1

doi: 10.1101/2024.12.31.630908

Figure Lengend Snippet: ( A-E ) WT, tax1bp1 / optn / ndp52 / nbr1 / p62 penta knockout (5 KO), optn / ndp52 / tax1bp1 triple knockout (TKO), and p62 KO HeLa cells were transiently transfected with or without MYC-UBB⁺¹. After 24 h, cells were given fresh DMEM for 16 h. Conditioned media and WCL were resolved by 15% SDS-PAGE and immunoblotted for UBB +1 . GAPDH was used as the loading control for WCL, and CLUSTERIN was used for media samples. ( F ) Representative immunofluorescence staining of WT and p62 KO HeLa cells expressing MYC-UBB⁺¹. Cells were fixed, permeabilized, and immunostained with a monoclonal antibody against UBB⁺¹ (green), and nuclei were stained with DAPI (blue). Scale bars: 5 μm. Statistical significance was determined by paired t-test, with data showing the mean ± SD from approximately 100 cells. ( G ) Representative immunoblot of UBB⁺¹ in insoluble fractions harvested from WT and p62 KO HeLa cells transfected with MYC-UBB⁺¹ plasmid. Cells were lysed with NP40-lysis buffer, clarified at 15,000 x g and the pellet resuspended in 1% SDS. Ponceau staining was used for loading control. Data represents the mean ± SD from at least three independent experiments. For all statistical tests, *, **, ***, p < 0.05, 0.01, and 0.001, respectively.

Article Snippet: SQSTM1/p62 with C-terminal HA tag (HA-SQSTM1/p62, 452 residues) was obtained from addgene (28027, deposited by Qing Zhong).

Techniques: Knock-Out, Triple Knockout, Transfection, SDS Page, Control, Immunofluorescence, Staining, Expressing, Western Blot, Plasmid Preparation, Lysis

Journal: bioRxiv

Article Title: Molecular mechanisms underlying p62-dependent secretion of the Alzheimer-associated ubiquitin variant, UBB +1

doi: 10.1101/2024.12.31.630908

Figure Lengend Snippet: (A) Immunofluorescence staining of endogenous SQSTM1/p62 in HeLa cells expressing MYC-UBB⁺¹. Cells were grown in complete media, fixed, permeabilized, and stained with monoclonal antibodies for UBB⁺¹ (green) and SQSTM1/p62 (red). Merged images show colocalization, with nuclei stained using DAPI (blue). Arrows point to small round bodies containing SQSTM1/p62 and UBB⁺¹. Scale bar: 5 μm. (B) Immunofluorescence staining of overexpressed HA-p62 in HeLa cells co-expressing MYC-UBB⁺¹. Cells were fixed, permeabilized, and stained with monoclonal antibodies for UBB⁺¹ (green) and HA (red). Merged images are shown, with nuclei counterstained using DAPI (blue). Scale bar: 2 μm. (C) Immunofluorescence staining of HeLa cells expressing HA-p62 and MYC-UBB⁺¹ F4A/I44A showed no clear staining of UBB⁺¹ F4A/I44A in p62-bodies. (D) Immunoblot analysis of conditioned media and WCL from HeLa cells comparing MYC-UBB⁺¹ and MYC-UBB⁺¹ F4A/I44A . 24 h after transfection followed by a 16 h incubation in fresh DMEM, proteins were resolved on 15% SDS-PAGE and detected using specific antibodies. GAPDH served as a loading control for WCL. (E) Co-immunoprecipitation (IP) of HA-p62 and MYC-UBB⁺¹ from HeLa cell lysates using either MYC- or FLAG-magnetic beads, respectively. Immunoprecipitates under non-denaturing conditions were probed by immunoblotting (IB) with anti-UBB⁺¹ and anti-HA antibodies. (F) Co-immunoprecipitation of endogenous SQSTM1/p62 with MYC-UBB⁺¹ from HeLa cells. Lysates were immunoprecipitated under non-denaturing conditions using an anti-SQSTM1/p62 antibody, resolved and probed with anti-p62 and anti-UBB⁺¹ antibodies. The antibody heavy chain is observed as a 50 kDa band (denoted by an asterisk). The anti-UBB⁺¹ antibodies detected both ubiquitinated and non-ubiquitinated forms of UBB⁺¹. (G) Co-immunoprecipitation of endogenous SQSTM1/p62 with MYC-UBB⁺¹ in HeLa cells treated with or without the E1 ubiquitin-activating enzyme inhibitor, TAK243 (1 μM, 3 h). Cell lysates were immunoprecipitated under non-denaturing conditions, and IB was performed using anti-SQSTM1/p62 and anti-UBB⁺¹ antibodies. The antibody heavy chain is detected as a 50 kDa band (denoted by an asterisk). (H) Immunofluorescence staining of HeLa cells co-expressing HA-p62 and MYC-UBB⁺¹ after 3 h of TAK243 treatment (same as in panel G). Cells were fixed, permeabilized, and stained with monoclonal antibodies for UBB⁺¹ (green) and HA (red). Merged images are shown, with nuclei counterstained using DAPI (blue). Scale bar: 2 μm.

Article Snippet: SQSTM1/p62 with C-terminal HA tag (HA-SQSTM1/p62, 452 residues) was obtained from addgene (28027, deposited by Qing Zhong).

Techniques: Immunofluorescence, Staining, Expressing, Bioprocessing, Western Blot, Transfection, Incubation, SDS Page, Control, Immunoprecipitation, Magnetic Beads, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: Molecular mechanisms underlying p62-dependent secretion of the Alzheimer-associated ubiquitin variant, UBB +1

doi: 10.1101/2024.12.31.630908

Figure Lengend Snippet: (A) Schematic representation of functional domain in full-length (FL) SQSTM1/p62, SQSTM1/p62ΔPB1 (lacking the PB1 domain), and SQSTM1/p62-LIR mutant (W340A/L343A). Immunoblot of overexpressed C-terminally HA-tagged SQSTM1/p62ΔPB1 and SQSTM1/p62 W340A/L343A . An asterisk (*) indicates the calculated molecular weights (MW) of the constructs. The calculated MW of the main isoform of FL SQSTM1/p62 is 47 kDa, yet complex covalent modifications result in an observed band migrating above 60 kDa. Likewise, the calculated MW of the truncated ΔPB1 construct is 35.5 kDa accompanied with a higher MW modified form. ( B and C ) HeLa cells co-expressing either HA-p62ΔPB1 and MYC-UBB +1 or HA-p62 W340A/L343A and MYC-UBB +1 was fixed and stained for HA (red) and UBB +1 (green) using respective monoclonal antibodies. Nuclei were counterstained with DAPI (blue). Scale bar: 5 μm. ( D-F ) WT and p62 KO HeLa cells co-expressing MYC-UBB +1 with HA-p62ΔPB1 ( D ), with HA-p62 W340A/L343A ( E ), or with HA-full-length (FL) p62 ( F ). After 24 h of transfection, cells were given fresh DMEM for 16 h. Conditioned media and WCL were separated on 10% or 15% SDS-PAGE, and indicated proteins were detected by immunoblotting using specific antibodies. GAPDH served as a loading control for WCL. Representative UBB +1 immunoblots are shown. Densitometric analysis of secreted UBB +1 intensity relative intensity in untreated cells was averaged from three independent experiments. Error bars represent means ± SD. Statistical significance: *, **, *** indicate p < 0.05, 0.01, and 0.001, respectively; ns = non-significant. ( G ) HeLa cells co-expressing the different SQSTM1/p62 species (as described in ) with MYC-UBB +1 was lysed, and cell lysates were subjected to immunoprecipitation (IP) using HA-magnetic beads. Elution was performed using HA peptides. Immunoprecipitated samples were immunoblotted (IB) with anti-HA and anti-UBB +1 antibodies.

Article Snippet: SQSTM1/p62 with C-terminal HA tag (HA-SQSTM1/p62, 452 residues) was obtained from addgene (28027, deposited by Qing Zhong).

Techniques: Functional Assay, Mutagenesis, Western Blot, Construct, Modification, Expressing, Staining, Bioprocessing, Transfection, SDS Page, Control, Immunoprecipitation, Magnetic Beads

Journal: bioRxiv

Article Title: Molecular mechanisms underlying p62-dependent secretion of the Alzheimer-associated ubiquitin variant, UBB +1

doi: 10.1101/2024.12.31.630908

Figure Lengend Snippet: SQSTM1/p62 critically affects both the intra- and extra-cellular fates of UBB +1 : (1) Autophagic clearance facilitated by SQSTM1/p62 and LC3, leading to either lysosomal degradation or extracellular expulsion via autophagosome-plasma membrane fusion mediated by SEC22B and STX4. (2) Sequestration into p62-induced round bodies when SQSTM1/p62 is elevated relative to levels of LC3. (3) Aggregation and intracellular retention in the absence – or insufficient levels – of SQSTM1/p62.

Article Snippet: SQSTM1/p62 with C-terminal HA tag (HA-SQSTM1/p62, 452 residues) was obtained from addgene (28027, deposited by Qing Zhong).

Techniques: Clinical Proteomics, Membrane