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mouse anti sqstm1 p62  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc mouse anti sqstm1 p62
    (A) Workflow of first proteomics experiments using the neddylation inhibitor MLN4924 to enrich unstable CRL targets (left) and cartoon representation of drug treatment mechanism (right). Cells were treated with MLN4924 (10 μM) or vehicle for 16 h. (B) Workflow of second proteomics experiments using tandem UBA domain fusions to SHKBP1 to enrich ubiquitinated substrates (left) and model of ligase trap (right). Cells stably expressing the corresponding construct were treated with MG-132 (20 μM) for 2 h. (C) Workflow of third proteomics experiments using the SHKBP1 F44A CUL3-binding deficient mutant to reduce CRL complex components in IP compared to SHKBP1 WT (left) and corresponding model (right). (D–F) Volcano plots from the three SILAC MS proteomics experiments, showing log 2 (fold changes of protein abundance in heavy/light samples) vs. statistical significance (−log 10 (p-value)). Proteins whose change was below the cutoff (fold change < 1.2) are indicated in grey. Those above the cutoff with p values above 0.05 are shown in green, and those with p values below 0.05 are shown in purple.
    Mouse Anti Sqstm1 P62, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 449 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mouse anti sqstm1 p62 - by Bioz Stars, 2026-10
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    Images

    1) Product Images from "Cullin-3 adaptor SHKBP1 inhibits SQSTM1/p62 oligomerization and Keap1 sequestration"

    Article Title: Cullin-3 adaptor SHKBP1 inhibits SQSTM1/p62 oligomerization and Keap1 sequestration

    Journal: The Journal of cell biology

    doi: 10.1083/jcb.202501207

    (A) Workflow of first proteomics experiments using the neddylation inhibitor MLN4924 to enrich unstable CRL targets (left) and cartoon representation of drug treatment mechanism (right). Cells were treated with MLN4924 (10 μM) or vehicle for 16 h. (B) Workflow of second proteomics experiments using tandem UBA domain fusions to SHKBP1 to enrich ubiquitinated substrates (left) and model of ligase trap (right). Cells stably expressing the corresponding construct were treated with MG-132 (20 μM) for 2 h. (C) Workflow of third proteomics experiments using the SHKBP1 F44A CUL3-binding deficient mutant to reduce CRL complex components in IP compared to SHKBP1 WT (left) and corresponding model (right). (D–F) Volcano plots from the three SILAC MS proteomics experiments, showing log 2 (fold changes of protein abundance in heavy/light samples) vs. statistical significance (−log 10 (p-value)). Proteins whose change was below the cutoff (fold change < 1.2) are indicated in grey. Those above the cutoff with p values above 0.05 are shown in green, and those with p values below 0.05 are shown in purple.
    Figure Legend Snippet: (A) Workflow of first proteomics experiments using the neddylation inhibitor MLN4924 to enrich unstable CRL targets (left) and cartoon representation of drug treatment mechanism (right). Cells were treated with MLN4924 (10 μM) or vehicle for 16 h. (B) Workflow of second proteomics experiments using tandem UBA domain fusions to SHKBP1 to enrich ubiquitinated substrates (left) and model of ligase trap (right). Cells stably expressing the corresponding construct were treated with MG-132 (20 μM) for 2 h. (C) Workflow of third proteomics experiments using the SHKBP1 F44A CUL3-binding deficient mutant to reduce CRL complex components in IP compared to SHKBP1 WT (left) and corresponding model (right). (D–F) Volcano plots from the three SILAC MS proteomics experiments, showing log 2 (fold changes of protein abundance in heavy/light samples) vs. statistical significance (−log 10 (p-value)). Proteins whose change was below the cutoff (fold change < 1.2) are indicated in grey. Those above the cutoff with p values above 0.05 are shown in green, and those with p values below 0.05 are shown in purple.

    Techniques Used: Stable Transfection, Expressing, Construct, Binding Assay, Mutagenesis, Multiplex sample analysis, Quantitative Proteomics

    (A) Western blot analysis of whole cell lysates (WCL) and α-GFP immunoprecipitates (IP) from HeLa cells co-transfected with HA-p62 and GFP-SHKBP1 or GFP empty vector (EV) as a control. (B) Western blot analysis of WCL and α-GFP IP from HeLa cells co-transfected with GFP-p62 and HA-SHKBP1 or HA empty vector (EV) as a control. (C and D) Western blot analysis of WCL and α-GFP IP to assess the interaction between SHKBP1 and exogenous (C) or endogenous p62 (D). HeLa cells were transfected with GFP-SHKBP1 in combination with HA-p62 (C) or alone (D), and treated with DMSO or the proteasome inhibitor MG-132 (20 μM) for 2 h or MLN4924 (10 μM) for 16 h. (E) HeLa cells were co-transfected with GFP-p62 and mScarlet-i-SHKBP1, treated with DMSO or the proteasome inhibitor MG-132 (20 μM) for 2 h or MLN4924 (10 μM) for 16 h, and then observed under confocal microscope 24 h post transfection. Scale bars: 10 μm.
    Figure Legend Snippet: (A) Western blot analysis of whole cell lysates (WCL) and α-GFP immunoprecipitates (IP) from HeLa cells co-transfected with HA-p62 and GFP-SHKBP1 or GFP empty vector (EV) as a control. (B) Western blot analysis of WCL and α-GFP IP from HeLa cells co-transfected with GFP-p62 and HA-SHKBP1 or HA empty vector (EV) as a control. (C and D) Western blot analysis of WCL and α-GFP IP to assess the interaction between SHKBP1 and exogenous (C) or endogenous p62 (D). HeLa cells were transfected with GFP-SHKBP1 in combination with HA-p62 (C) or alone (D), and treated with DMSO or the proteasome inhibitor MG-132 (20 μM) for 2 h or MLN4924 (10 μM) for 16 h. (E) HeLa cells were co-transfected with GFP-p62 and mScarlet-i-SHKBP1, treated with DMSO or the proteasome inhibitor MG-132 (20 μM) for 2 h or MLN4924 (10 μM) for 16 h, and then observed under confocal microscope 24 h post transfection. Scale bars: 10 μm.

    Techniques Used: Western Blot, Transfection, Plasmid Preparation, Control, Microscopy

    (A) Domain map of SHKBP1 and truncations used in this paper. (B) Representative live-cell images showing localization of full-length (FL) and truncated forms of SHKBP1. HeLa cells were transfected with the indicated GFP-tagged SHKBP1 construct and observed by confocal microscopy 24 h post-transfection. Scale bars: 10 μm. (C) Western blot analysis of α-GFP IP of lysates from HeLa cells co-transfected with HA-CUL3 and either the indicated GFP-SHKBP1 truncation or GFP empty vector (EV) as a control. (D) Western blot analysis of α-GFP IP of lysates from HeLa cells co-transfected with HA-p62 and either the indicated GFP-SHKBP1 truncation or GFP-EV as a control. (E) Domain map of p62 and truncations used in this paper. (F) Representative live cell images showing localization of p62 truncations. HeLa cells were transfected with the indicated GFP-tagged p62 construct and observed by confocal microscopy 24 h post-transfection. Scale bars: 10 μm. (G) Western blot analysis of α-GFP IP of lysates from HeLa cells co-transfected with HA-SHKBP1 and the indicated GFP-p62 construct. (H) AlphaFold3 structural prediction of the interaction between SHKBP1-WD domain (magenta) and p62 (green). Electrostatic interactions (blue) indicated between p62 residues K7, R18, D92, D93 and R96 and SHKBP1 residues R357, K359, D360, D362, and E367 (pink), respectively. ipTM = 0.73, pTM = 0.54. (I) Western blot analysis of α-GFP IP of lysates from HeLa cells co-transfected with the indicated FLAG-SHKBP1 and GFP-p62 constructs. (J) Quantification of FLAG-SHKBP1 from GFP IP samples to assess the strength of interaction between p62 and SHKBP1, with band intensities normalized to FLAG-SHKBP1 levels in WCL and to GFP-p62 levels in IP (n = 3). Exact p values indicated from two-way ANOVA with Tukey’s post-hoc test. (K) Representative live cell images showing localization of p62 point mutants. HeLa cells were transfected with the indicated GFP-tagged p62 construct and observed by confocal microscopy 24 h post transfection. Scale bars: 10 μm.
    Figure Legend Snippet: (A) Domain map of SHKBP1 and truncations used in this paper. (B) Representative live-cell images showing localization of full-length (FL) and truncated forms of SHKBP1. HeLa cells were transfected with the indicated GFP-tagged SHKBP1 construct and observed by confocal microscopy 24 h post-transfection. Scale bars: 10 μm. (C) Western blot analysis of α-GFP IP of lysates from HeLa cells co-transfected with HA-CUL3 and either the indicated GFP-SHKBP1 truncation or GFP empty vector (EV) as a control. (D) Western blot analysis of α-GFP IP of lysates from HeLa cells co-transfected with HA-p62 and either the indicated GFP-SHKBP1 truncation or GFP-EV as a control. (E) Domain map of p62 and truncations used in this paper. (F) Representative live cell images showing localization of p62 truncations. HeLa cells were transfected with the indicated GFP-tagged p62 construct and observed by confocal microscopy 24 h post-transfection. Scale bars: 10 μm. (G) Western blot analysis of α-GFP IP of lysates from HeLa cells co-transfected with HA-SHKBP1 and the indicated GFP-p62 construct. (H) AlphaFold3 structural prediction of the interaction between SHKBP1-WD domain (magenta) and p62 (green). Electrostatic interactions (blue) indicated between p62 residues K7, R18, D92, D93 and R96 and SHKBP1 residues R357, K359, D360, D362, and E367 (pink), respectively. ipTM = 0.73, pTM = 0.54. (I) Western blot analysis of α-GFP IP of lysates from HeLa cells co-transfected with the indicated FLAG-SHKBP1 and GFP-p62 constructs. (J) Quantification of FLAG-SHKBP1 from GFP IP samples to assess the strength of interaction between p62 and SHKBP1, with band intensities normalized to FLAG-SHKBP1 levels in WCL and to GFP-p62 levels in IP (n = 3). Exact p values indicated from two-way ANOVA with Tukey’s post-hoc test. (K) Representative live cell images showing localization of p62 point mutants. HeLa cells were transfected with the indicated GFP-tagged p62 construct and observed by confocal microscopy 24 h post transfection. Scale bars: 10 μm.

    Techniques Used: Transfection, Construct, Confocal Microscopy, Western Blot, Plasmid Preparation, Control, Structural Proteomics

    (A) Western blot analysis of in vivo ubiquitination assay. WT or SHKBP1 KO HeLa cells were co-transfected with His-ubiquitin and either GFP-p62 or GFP EV, and overexpressed (OE) group represents WT cells that were also co-transfected with FLAG-SHKBP1. 24 h post transfection, cells were treated with MG-132 (20 μM) for 2 h and subjected to His pull-down using Ni-NTA agarose and Western blot. (B) Western blot analysis of the whole-cell lysates (WCL) from WT, SHKBP1 KO, and FLAG-SHKBP1 overexpressing (OE) HeLa cells. (C) Representative live cell images showing p62 body formation. WT or SHKBP1 KO HeLa cells were transfected with GFP-p62 either alone (left) or in combination with mScarlet-i-SHKBP1 (right, where OE refers to SHKBP1 overexpression in WT cells and RE (rescue) refers to SHKBP1 overexpression in KO cells) and imaged using confocal microscopy 24 h post-transfection. Scale bars: 20 μm. (D) Quantification of the average p62 body size of images shown in (C) from three independently plated samples. n = 51 (KO), 43 (WT), 37 (OE), and 35 (RE). Exact p values indicated (****, p < 0.0001) from one-way ANOVA with Tukey’s post-hoc test. (E) Western blot analysis of WCL from WT or SHKBP1 KO HeLa cells after DSP crosslinking. Cells were treated with MG-132 (0.5 μM) for 12 h, crosslinked with 0.4 mg/mL DSP at 4 °C for 2 h, and lysed in IP lysis buffer with 1% SDS. The lysates mixed with reducing or nonreducing loading buffer (i.e., with or without β-mercaptoethanol) and were analyzed by Western blot. (F) Quantification of the ratio of intensities of monomeric to total p62. Intensities were normalized to the WT without MG-132 treatment group. n = 6. Exact p values indicated (****, p < 0.0001) from two-way ANOVA.
    Figure Legend Snippet: (A) Western blot analysis of in vivo ubiquitination assay. WT or SHKBP1 KO HeLa cells were co-transfected with His-ubiquitin and either GFP-p62 or GFP EV, and overexpressed (OE) group represents WT cells that were also co-transfected with FLAG-SHKBP1. 24 h post transfection, cells were treated with MG-132 (20 μM) for 2 h and subjected to His pull-down using Ni-NTA agarose and Western blot. (B) Western blot analysis of the whole-cell lysates (WCL) from WT, SHKBP1 KO, and FLAG-SHKBP1 overexpressing (OE) HeLa cells. (C) Representative live cell images showing p62 body formation. WT or SHKBP1 KO HeLa cells were transfected with GFP-p62 either alone (left) or in combination with mScarlet-i-SHKBP1 (right, where OE refers to SHKBP1 overexpression in WT cells and RE (rescue) refers to SHKBP1 overexpression in KO cells) and imaged using confocal microscopy 24 h post-transfection. Scale bars: 20 μm. (D) Quantification of the average p62 body size of images shown in (C) from three independently plated samples. n = 51 (KO), 43 (WT), 37 (OE), and 35 (RE). Exact p values indicated (****, p < 0.0001) from one-way ANOVA with Tukey’s post-hoc test. (E) Western blot analysis of WCL from WT or SHKBP1 KO HeLa cells after DSP crosslinking. Cells were treated with MG-132 (0.5 μM) for 12 h, crosslinked with 0.4 mg/mL DSP at 4 °C for 2 h, and lysed in IP lysis buffer with 1% SDS. The lysates mixed with reducing or nonreducing loading buffer (i.e., with or without β-mercaptoethanol) and were analyzed by Western blot. (F) Quantification of the ratio of intensities of monomeric to total p62. Intensities were normalized to the WT without MG-132 treatment group. n = 6. Exact p values indicated (****, p < 0.0001) from two-way ANOVA.

    Techniques Used: Ubiquitin Proteomics, Western Blot, In Vivo, Transfection, Over Expression, Confocal Microscopy, Lysis

    (A) WT and SHKBP1 KO HeLa cells were transfected with GFP-p62 alone or in combination with mScarlet-i-SHKBP1. 24 h later, cells were subjected to live-cell imaging by confocal microscopy and analysis by fluorescence recovery after photobleaching (FRAP). Shown is a representative image for each condition. A pre-bleach image is provided along with a timecourse of post-bleach images (post-bleach time indicated in min). Scale bars: 2 μm. (B) Quantification of the fluorescence recovery rates of GFP-p62 from FRAP experiments. Data were analyzed with GraphPad Prism using nonlinear regression (curve fit), shown as mean ± standard deviation (SD). n = 3 for each group from three independent experiments. (C) Quantification of the half-life of fluorescence recovery of GFP-p62 from FRAP experiments. n = 20 for each group from three independent experiments. Exact p values indicated from one-way ANOVA with Tukey’s post-hoc test. (D) Western blot analysis of the whole-cell lysates from three HEK 293T cell lines stably expressing endogenously tagged p62 using mNeonGreen 2 (mNG): SHKBP1 WT, SHKBP1 KO, and SHKBP1 WT with transient overexpression (OE) of a large amount of FLAG-SHKBP1. The hollow arrowhead indicates the FLAG-SHKBP1, and the solid arrowhead indicates untagged (endogenous) SHKBP1. (E) Western blot analysis of WCL, flow-through (FT), and anti-mNeonGreen IP to assess the interaction between SHKBP1 and endogenously tagged p62 in a HEK293T cell line stably expressing mNG-p62 and transfected with a small amount of FLAG-SHKBP1. For the p62 blot, asterisks denote background bands from p62 antibody and solid arrowhead indicates mNG11-tagged p62. For the SHKBP1 blot, hollow arrowhead indicates the FLAG-SHKBP1, and solid arrowhead indicates untagged (endogenous) SHKBP1. (F) Representative live cell images showing single particle tracking of endogenous mNG-p62 by confocal microscopy. HEK 293T cell lines stably expressing mNG-p62 were transfected with either mScarlet-i (in both SHKBP1 KO and SHKBP1 WT backgrounds) or mScarlet-i-SHKBP1 (in SHKBP1 WT background, “OE”). Tracked p62 bodies are indicated by magenta circles, and movement tracks are shown by colored lines. Scale bars: 5 μm. (G) Quantification of the mean speed of tracked p62 bodies in SHKBP1 KO, WT, and OE conditions. n = 200–300 p62 bodies collected from three independently plated samples. Exact p values indicated from one-way ANOVA with Tukey’s post-hoc test. (H) Representative live cell images showing endogenous p62 body shapes. HEK 293T stable cell lines were transfected with the indicated mScarlet-i constructs and observed by confocal microscopy 24 h post-transfection. Scale bars: 5 μm. (I–L) Quantification of the mNG p62 body roundness, solidity, circularity and aspect ratio. n = 1000–1300 p62 bodies collected from three independently plated samples. Exact p values indicated from one-way ANOVA with Tukey’s post-hoc test.
    Figure Legend Snippet: (A) WT and SHKBP1 KO HeLa cells were transfected with GFP-p62 alone or in combination with mScarlet-i-SHKBP1. 24 h later, cells were subjected to live-cell imaging by confocal microscopy and analysis by fluorescence recovery after photobleaching (FRAP). Shown is a representative image for each condition. A pre-bleach image is provided along with a timecourse of post-bleach images (post-bleach time indicated in min). Scale bars: 2 μm. (B) Quantification of the fluorescence recovery rates of GFP-p62 from FRAP experiments. Data were analyzed with GraphPad Prism using nonlinear regression (curve fit), shown as mean ± standard deviation (SD). n = 3 for each group from three independent experiments. (C) Quantification of the half-life of fluorescence recovery of GFP-p62 from FRAP experiments. n = 20 for each group from three independent experiments. Exact p values indicated from one-way ANOVA with Tukey’s post-hoc test. (D) Western blot analysis of the whole-cell lysates from three HEK 293T cell lines stably expressing endogenously tagged p62 using mNeonGreen 2 (mNG): SHKBP1 WT, SHKBP1 KO, and SHKBP1 WT with transient overexpression (OE) of a large amount of FLAG-SHKBP1. The hollow arrowhead indicates the FLAG-SHKBP1, and the solid arrowhead indicates untagged (endogenous) SHKBP1. (E) Western blot analysis of WCL, flow-through (FT), and anti-mNeonGreen IP to assess the interaction between SHKBP1 and endogenously tagged p62 in a HEK293T cell line stably expressing mNG-p62 and transfected with a small amount of FLAG-SHKBP1. For the p62 blot, asterisks denote background bands from p62 antibody and solid arrowhead indicates mNG11-tagged p62. For the SHKBP1 blot, hollow arrowhead indicates the FLAG-SHKBP1, and solid arrowhead indicates untagged (endogenous) SHKBP1. (F) Representative live cell images showing single particle tracking of endogenous mNG-p62 by confocal microscopy. HEK 293T cell lines stably expressing mNG-p62 were transfected with either mScarlet-i (in both SHKBP1 KO and SHKBP1 WT backgrounds) or mScarlet-i-SHKBP1 (in SHKBP1 WT background, “OE”). Tracked p62 bodies are indicated by magenta circles, and movement tracks are shown by colored lines. Scale bars: 5 μm. (G) Quantification of the mean speed of tracked p62 bodies in SHKBP1 KO, WT, and OE conditions. n = 200–300 p62 bodies collected from three independently plated samples. Exact p values indicated from one-way ANOVA with Tukey’s post-hoc test. (H) Representative live cell images showing endogenous p62 body shapes. HEK 293T stable cell lines were transfected with the indicated mScarlet-i constructs and observed by confocal microscopy 24 h post-transfection. Scale bars: 5 μm. (I–L) Quantification of the mNG p62 body roundness, solidity, circularity and aspect ratio. n = 1000–1300 p62 bodies collected from three independently plated samples. Exact p values indicated from one-way ANOVA with Tukey’s post-hoc test.

    Techniques Used: Transfection, Live Cell Imaging, Confocal Microscopy, Fluorescence, Standard Deviation, Western Blot, Stable Transfection, Expressing, Over Expression, Single-particle Tracking, Construct

    (A) Western blot analysis of co-IP experiments for endogenous p62. Lysates were from WT HeLa cells, SHKBP1 KO HeLa cells, and HeLa cells transfected with mScarlet-i-SHKBP1 (OE). (B) Quantification of Keap1 from p62-IP to demonstrate the interaction between endogenous p62 and Keap1, with band intensities normalized to actin levels (n = 3). Exact p values indicated from one-way ANOVA with Tukey’s post-hoc test. C) Western blot analysis of WCL from WT and SHKBP1 KO HeLa cells treated with As(III) (10 μM) for the indicated times. (D) Quantification of Keap1 protein levels, with band intensities normalized to actin levels (n = 5). Exact p values indicated (****, p < 0.0001) from two-way ANOVA. (E) Confocal microscopy analysis of WT and KO SHKBP1 HeLa cells co-transfected with GFP-p62 and miRFP-Keap1 and treated with As(III) (10 μM) for 4 h or with DMSO as control. Scale bars: 20 μm. (F and G) Quantification of Keap1 aggregate size (F) and p62 body size (G) in WT and SHKBP1 KO HeLa cells after As(III) treatment (representative images shown in 6E). n = 11 images from three independently plated samples. Exact p values indicated from unpaired two-tailed Student’s t test. (H) Confocal microscopy analysis of HeLa cells co-transfected with GFP-p62 and miRFP-Keap1, with or without mScarlet-i-SHKBP1, and treated with As(III) (10 μM) for 4 h or with DMSO as control. Scale bars: 20 μm. (I and J) Quantification of p62 body size (I) and the percentage of cells containing Keap1 aggregates (J) in control of SHKBP1-overexpressing (OE) HeLa cells with or without As(III) treatment (representative images shown in 6H). n = 8–12 images from three independently plated samples. Exact p values indicated from two-way ANOVA. (K) Immunofluorescence (IF) analysis of endogenous p62 and Keap1 in WT and SHKBP1 KO HeLa cells by confocal microscopy. Cells were treated with As(III) (10 μM) for indicated times. Scale bars: 10 μm; 5 μm (zoomed-in images). (L–N) Quantification of Keap1 aggregate size (L), p62 body size (M), and colocalization of Keap1 and p62 (N) in WT and SHKBP1 KO HeLa cells with As(III) treatment for the indicated times (representative images shown in 6K). n = 13–18 images from three independently plated samples. Exact p values indicated (****, p < 0.0001) from two-way ANOVA with Tukey’s post-hoc test.
    Figure Legend Snippet: (A) Western blot analysis of co-IP experiments for endogenous p62. Lysates were from WT HeLa cells, SHKBP1 KO HeLa cells, and HeLa cells transfected with mScarlet-i-SHKBP1 (OE). (B) Quantification of Keap1 from p62-IP to demonstrate the interaction between endogenous p62 and Keap1, with band intensities normalized to actin levels (n = 3). Exact p values indicated from one-way ANOVA with Tukey’s post-hoc test. C) Western blot analysis of WCL from WT and SHKBP1 KO HeLa cells treated with As(III) (10 μM) for the indicated times. (D) Quantification of Keap1 protein levels, with band intensities normalized to actin levels (n = 5). Exact p values indicated (****, p < 0.0001) from two-way ANOVA. (E) Confocal microscopy analysis of WT and KO SHKBP1 HeLa cells co-transfected with GFP-p62 and miRFP-Keap1 and treated with As(III) (10 μM) for 4 h or with DMSO as control. Scale bars: 20 μm. (F and G) Quantification of Keap1 aggregate size (F) and p62 body size (G) in WT and SHKBP1 KO HeLa cells after As(III) treatment (representative images shown in 6E). n = 11 images from three independently plated samples. Exact p values indicated from unpaired two-tailed Student’s t test. (H) Confocal microscopy analysis of HeLa cells co-transfected with GFP-p62 and miRFP-Keap1, with or without mScarlet-i-SHKBP1, and treated with As(III) (10 μM) for 4 h or with DMSO as control. Scale bars: 20 μm. (I and J) Quantification of p62 body size (I) and the percentage of cells containing Keap1 aggregates (J) in control of SHKBP1-overexpressing (OE) HeLa cells with or without As(III) treatment (representative images shown in 6H). n = 8–12 images from three independently plated samples. Exact p values indicated from two-way ANOVA. (K) Immunofluorescence (IF) analysis of endogenous p62 and Keap1 in WT and SHKBP1 KO HeLa cells by confocal microscopy. Cells were treated with As(III) (10 μM) for indicated times. Scale bars: 10 μm; 5 μm (zoomed-in images). (L–N) Quantification of Keap1 aggregate size (L), p62 body size (M), and colocalization of Keap1 and p62 (N) in WT and SHKBP1 KO HeLa cells with As(III) treatment for the indicated times (representative images shown in 6K). n = 13–18 images from three independently plated samples. Exact p values indicated (****, p < 0.0001) from two-way ANOVA with Tukey’s post-hoc test.

    Techniques Used: Western Blot, Co-Immunoprecipitation Assay, Transfection, Confocal Microscopy, Control, Two Tailed Test, Immunofluorescence

    (A) IF analysis of endogenous Nrf2 in WT and SHKBP1 KO HeLa cells by confocal microscopy. Nuclei were stained with DAPI (magenta). Cells were treated with As(III) (10 μM) for the indicated times. Scale bars: 15 μm. (B) Quantification of nuclear levels of Nrf2 in WT and SHKBP1 KO HeLa cells with As(III) treatment for the indicated times (representative images shown in (A). n = 9 images each including ~30 cells from three independently plated samples. Exact p values indicated (****, p < 0.0001) from two-way ANOVA with Tukey’s post-hoc test. (C) Western blot analysis of cytosolic and nuclear fractions from WT and SHKBP1 KO HeLa cells treated with As(III) (10 μM) for the indicated times. Tubulin and Histone H3 are used to qualitatively assess the purity of each fraction. (D) Quantification of nuclear Nrf2 protein levels, with band intensities normalized to Histone H3 levels (n = 3). Exact p values indicated from two-way ANOVA. (E) Role of SHKBP1 in p62-Keap1-Nrf2 pathway.
    Figure Legend Snippet: (A) IF analysis of endogenous Nrf2 in WT and SHKBP1 KO HeLa cells by confocal microscopy. Nuclei were stained with DAPI (magenta). Cells were treated with As(III) (10 μM) for the indicated times. Scale bars: 15 μm. (B) Quantification of nuclear levels of Nrf2 in WT and SHKBP1 KO HeLa cells with As(III) treatment for the indicated times (representative images shown in (A). n = 9 images each including ~30 cells from three independently plated samples. Exact p values indicated (****, p < 0.0001) from two-way ANOVA with Tukey’s post-hoc test. (C) Western blot analysis of cytosolic and nuclear fractions from WT and SHKBP1 KO HeLa cells treated with As(III) (10 μM) for the indicated times. Tubulin and Histone H3 are used to qualitatively assess the purity of each fraction. (D) Quantification of nuclear Nrf2 protein levels, with band intensities normalized to Histone H3 levels (n = 3). Exact p values indicated from two-way ANOVA. (E) Role of SHKBP1 in p62-Keap1-Nrf2 pathway.

    Techniques Used: Knock-Out, Translocation Assay, Confocal Microscopy, Staining, Western Blot

    Related Articles

    Western Blot:

    Article Title: Molecular mechanisms underlying p62-dependent secretion of the Alzheimer-associated ubiquitin variant UBB +1 .
    Article Snippet: Rabbit anti- LC3 (WB: 1:2,000, 51520) was from Abcam. .. Mouse anti- SQSTM1/p62 (WB 1:1,000, IF, 1:250 88588), mouse anti- STX4 (WB 1:1,000, E6W7B), mouse anti- STX17 (WB 1:1,000, D3D7H), rabbit anti- cleaved caspase 3 (WB 1:1,000, 9661) were from Cell Signaling Technology. .. Rabbit anti- STX3 (WB 1:1,000, Synaptic systems, 110032).

    Article Title: Inhibition of SQSTM1/p62 oligomerization and Keap1 sequestration by the Cullin-3 adaptor SHKBP1
    Article Snippet: .. The following antibodies were used for Western blot: mouse anti-SQSTM1/p62 (D5L7G), Cell Signaling Technology (88588); rabbit anti SQSTM1/p62, ABclonal (A19700); mouse anti-Keap1, Santa Cruz Biotechnology (sc-365626); rabbit anti-SHKBP1, Invitrogen (PA521731); mouse anti-Ubiquitin, Santa Cruz Biotechnology (sc-8017); mouse anti-GFP, Santa Cruz Biotechnology (sc-9996); mouse anti-mCherry, Sigma-Aldrich (MAB131873); mouse anti-Actin, MP Bio (08691001); rabbit anti-FLAG, Sigma-Aldrich (F7425); mouse anti-HA, Covance (MMS-101R); The following antibodies were used for IF: rabbit anti-FLAG, Sigma-Aldrich (F7425); rabbit anti SQSTM1/p62, ABclonal (A19700); mouse anti-Keap1, Santa Cruz Biotechnology (sc-365626); goat anti-mouse HRP, Jackson ImmunoResearch Labs (115-035-146); goat anti-rabbit HRP, Jackson ImmunoResearch Labs (111-035-144). .. Reagents were obtained from the following sources: DSP (Dithiobis(succinimidyl propionate)), TCI America (TCD2473); MLN4924, Cayman (15217); MG-132, Selleckchem (S2619); GFP-Trap magnetic agarose, ChromoTek (gtma-20); Protein G Sepharose, BioVision (6511); Ni-NTA agarose, QIAGEN (133203974); Anti-HA magnetic beads, Thermo Scientific (88836); Anti-FLAG magnetic beads, made in-house from NHS magnetic beads, Nacalai USA (TAS8848N1141-10MG) and Anti DYKDDDDK tag, Fujifilm (012-22383); Lipofectamine 2000, Invitrogen (11668019); RNase A, Research Products International (R21750); Puromycin dihydrochloride, Sigma-Aldrich (P8833); Blasticidin S hydrochloride, 10 mg/ml in HEPES buffer, Alfa Aesar (J67216); cOmplete Protease Inhibitor Cocktail, Roche (5056489001); ProLong Diamond Antifade Mountant with DAPI, Thermo Fisher (P36971); Clarity Western ECL Substrate, Bio-Rad (1705061).

    Article Title: Molecular mechanisms underlying p62-dependent secretion of the Alzheimer-associated ubiquitin variant, UBB +1
    Article Snippet: Rabbit anti-LC3 (WB: 1:2000, 51520) was from Abcam. .. Mouse anti-SQSTM1/p62 (WB 1:1000, IF, 1:250 88588), mouse anti-STX4 (WB 1:1000, E6W7B), mouse anti-STX17 (WB 1:1000, D3D7H) were from Cell Signaling Technology. .. Rabbit anti-STX3 (WB 1:1000, Synaptic systems, 110032).

    Article Title: Molecular mechanisms underlying p62-dependent secretion of the Alzheimer-associated ubiquitin variant UBB +1
    Article Snippet: Rabbit anti-LC3 (WB: 1:2,000, 51520) was from Abcam. .. Mouse anti-SQSTM1/p62 (WB 1:1,000, IF, 1:250 88588), mouse anti-STX4 (WB 1:1,000, E6W7B), mouse anti-STX17 (WB 1:1,000, D3D7H), rabbit anti-cleaved caspase 3 (WB 1:1,000, 9661) were from Cell Signaling Technology. .. Rabbit anti-STX3 (WB 1:1,000, Synaptic systems, 110032).

    Article Title: Cullin-3 adaptor SHKBP1 inhibits SQSTM1/p62 oligomerization and Keap1 sequestration
    Article Snippet: .. The following antibodies were used for Western blot: mouse anti-SQSTM1/p62 (D5L7G), Cell Signaling Technology (88588, RRID:AB_2800125); rabbit anti SQSTM1/p62, ABclonal (A19700, RRID:AB_2862742); mouse anti-Keap1, Santa Cruz Biotechnology (sc-365626, RRID:AB_10844829); rabbit anti-SHKBP1, Bethyl Laboratories (A304–891, RRID:AB_2621086); mouse anti-SHKBP1, Santa Cruz Biotechnology (sc-390121, RRID unavailable); mouse anti-ubiquitin, Santa Cruz Biotechnology (sc-8017, RRID:AB_628423); mouse anti-GFP, Santa Cruz Biotechnology (sc-9996, RRID:AB_627695); mouse anti-mCherry, Sigma-Aldrich (MAB131873, RRID:AB_11034849); mouse anti-Actin, MP Bio (08691001, RRID:AB_2335127); rabbit anti-FLAG, Sigma-Aldrich (F7425, RRID:AB_439687); mouse anti-HA, Covance (MMS-101R, RRID:AB_291262); rabbit anti-Tubulin, Cell Signaling Technology (2128S, RRID:AB_823664); rabbit anti-Histone H3, Cell Signaling Technology (4499S, RRID:AB_10544537); mouse anti-Nrf2 (A-10), Santa Cruz Biotechnology (sc-365949, RRID:AB_ 10917561). .. The following antibodies were used for IF: rabbit anti-FLAG, Sigma-Aldrich (F7425, RRID:AB_439687); rabbit anti-LC3A/B (D3U4C), Cell Signaling Technology (12741S, RRID:AB_2617131); rabbit anti SQSTM1/p62, ABclonal (A19700, RRID:AB_2862742); mouse anti-Keap1, Santa Cruz Biotechnology (sc-365626, RRID:AB_10844829); mouse anti-Nrf2 (D1Z9C), Cell Signaling Technology (12721S, RRID:AB_ 2715528); Alexa Fluor 594-conjugated goat anti-mouse IgG, Thermo Fisher ( R37121 , RRID:AB_2556549); Alexa Fluor 488-conjugated goat anti-rabbit IgG, Thermo Fisher (A11008, RRID:AB_ 143165); goat anti-mouse HRP, Jackson ImmunoResearch Labs (115–035-146, RRID:AB_2307392); goat anti-rabbit HRP, Jackson ImmunoResearch Labs (111–035-144, RRID:AB_2307391).

    Blocking Assay:

    Article Title: Impact of endoplasmic reticulum aminopeptidases 1 (ERAP1) and 2 (ERAP2) on neutrophil cellular functions
    Article Snippet: .. 24 h post-rhERAP administration, 2.5 × 10 5 neutrophils were washed in PBS and fixed with 4% PFA for 10 min at RT, followed by permeabilization with 0.1% Triton X-100 in PBS for 10 min at RT, blocking in 5% BSA in PBS for 1 h at RT, and incubation for overnight at 4°C in an humified chamber with primary antibodies Rabbit anti-LC3B, 1:1500 (Cell Signalling); Mouse anti-SQSTM1-P62, 1:300 (Cell Signalling), prepared in 1% BSA-PBS. .. The next day, samples were washed three times with PBS and incubated for 45 min at RT with secondary antibodies (Goat anti-mouse Alexa Fluor 488 (ab150113) or 647 (ab150115), or Goat anti-rabbit Alexa Fluor 488,647 (ab150079), 1:500, abcam, Cambridge, United Kingdom) prepared in 1% BSA-PBS.

    Incubation:

    Article Title: Impact of endoplasmic reticulum aminopeptidases 1 (ERAP1) and 2 (ERAP2) on neutrophil cellular functions
    Article Snippet: .. 24 h post-rhERAP administration, 2.5 × 10 5 neutrophils were washed in PBS and fixed with 4% PFA for 10 min at RT, followed by permeabilization with 0.1% Triton X-100 in PBS for 10 min at RT, blocking in 5% BSA in PBS for 1 h at RT, and incubation for overnight at 4°C in an humified chamber with primary antibodies Rabbit anti-LC3B, 1:1500 (Cell Signalling); Mouse anti-SQSTM1-P62, 1:300 (Cell Signalling), prepared in 1% BSA-PBS. .. The next day, samples were washed three times with PBS and incubated for 45 min at RT with secondary antibodies (Goat anti-mouse Alexa Fluor 488 (ab150113) or 647 (ab150115), or Goat anti-rabbit Alexa Fluor 488,647 (ab150079), 1:500, abcam, Cambridge, United Kingdom) prepared in 1% BSA-PBS.



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    Image Search Results


    (A) Workflow of first proteomics experiments using the neddylation inhibitor MLN4924 to enrich unstable CRL targets (left) and cartoon representation of drug treatment mechanism (right). Cells were treated with MLN4924 (10 μM) or vehicle for 16 h. (B) Workflow of second proteomics experiments using tandem UBA domain fusions to SHKBP1 to enrich ubiquitinated substrates (left) and model of ligase trap (right). Cells stably expressing the corresponding construct were treated with MG-132 (20 μM) for 2 h. (C) Workflow of third proteomics experiments using the SHKBP1 F44A CUL3-binding deficient mutant to reduce CRL complex components in IP compared to SHKBP1 WT (left) and corresponding model (right). (D–F) Volcano plots from the three SILAC MS proteomics experiments, showing log 2 (fold changes of protein abundance in heavy/light samples) vs. statistical significance (−log 10 (p-value)). Proteins whose change was below the cutoff (fold change < 1.2) are indicated in grey. Those above the cutoff with p values above 0.05 are shown in green, and those with p values below 0.05 are shown in purple.

    Journal: The Journal of cell biology

    Article Title: Cullin-3 adaptor SHKBP1 inhibits SQSTM1/p62 oligomerization and Keap1 sequestration

    doi: 10.1083/jcb.202501207

    Figure Lengend Snippet: (A) Workflow of first proteomics experiments using the neddylation inhibitor MLN4924 to enrich unstable CRL targets (left) and cartoon representation of drug treatment mechanism (right). Cells were treated with MLN4924 (10 μM) or vehicle for 16 h. (B) Workflow of second proteomics experiments using tandem UBA domain fusions to SHKBP1 to enrich ubiquitinated substrates (left) and model of ligase trap (right). Cells stably expressing the corresponding construct were treated with MG-132 (20 μM) for 2 h. (C) Workflow of third proteomics experiments using the SHKBP1 F44A CUL3-binding deficient mutant to reduce CRL complex components in IP compared to SHKBP1 WT (left) and corresponding model (right). (D–F) Volcano plots from the three SILAC MS proteomics experiments, showing log 2 (fold changes of protein abundance in heavy/light samples) vs. statistical significance (−log 10 (p-value)). Proteins whose change was below the cutoff (fold change < 1.2) are indicated in grey. Those above the cutoff with p values above 0.05 are shown in green, and those with p values below 0.05 are shown in purple.

    Article Snippet: The following antibodies were used for Western blot: mouse anti-SQSTM1/p62 (D5L7G), Cell Signaling Technology (88588, RRID:AB_2800125); rabbit anti SQSTM1/p62, ABclonal (A19700, RRID:AB_2862742); mouse anti-Keap1, Santa Cruz Biotechnology (sc-365626, RRID:AB_10844829); rabbit anti-SHKBP1, Bethyl Laboratories (A304–891, RRID:AB_2621086); mouse anti-SHKBP1, Santa Cruz Biotechnology (sc-390121, RRID unavailable); mouse anti-ubiquitin, Santa Cruz Biotechnology (sc-8017, RRID:AB_628423); mouse anti-GFP, Santa Cruz Biotechnology (sc-9996, RRID:AB_627695); mouse anti-mCherry, Sigma-Aldrich (MAB131873, RRID:AB_11034849); mouse anti-Actin, MP Bio (08691001, RRID:AB_2335127); rabbit anti-FLAG, Sigma-Aldrich (F7425, RRID:AB_439687); mouse anti-HA, Covance (MMS-101R, RRID:AB_291262); rabbit anti-Tubulin, Cell Signaling Technology (2128S, RRID:AB_823664); rabbit anti-Histone H3, Cell Signaling Technology (4499S, RRID:AB_10544537); mouse anti-Nrf2 (A-10), Santa Cruz Biotechnology (sc-365949, RRID:AB_ 10917561).

    Techniques: Stable Transfection, Expressing, Construct, Binding Assay, Mutagenesis, Multiplex sample analysis, Quantitative Proteomics

    (A) Western blot analysis of whole cell lysates (WCL) and α-GFP immunoprecipitates (IP) from HeLa cells co-transfected with HA-p62 and GFP-SHKBP1 or GFP empty vector (EV) as a control. (B) Western blot analysis of WCL and α-GFP IP from HeLa cells co-transfected with GFP-p62 and HA-SHKBP1 or HA empty vector (EV) as a control. (C and D) Western blot analysis of WCL and α-GFP IP to assess the interaction between SHKBP1 and exogenous (C) or endogenous p62 (D). HeLa cells were transfected with GFP-SHKBP1 in combination with HA-p62 (C) or alone (D), and treated with DMSO or the proteasome inhibitor MG-132 (20 μM) for 2 h or MLN4924 (10 μM) for 16 h. (E) HeLa cells were co-transfected with GFP-p62 and mScarlet-i-SHKBP1, treated with DMSO or the proteasome inhibitor MG-132 (20 μM) for 2 h or MLN4924 (10 μM) for 16 h, and then observed under confocal microscope 24 h post transfection. Scale bars: 10 μm.

    Journal: The Journal of cell biology

    Article Title: Cullin-3 adaptor SHKBP1 inhibits SQSTM1/p62 oligomerization and Keap1 sequestration

    doi: 10.1083/jcb.202501207

    Figure Lengend Snippet: (A) Western blot analysis of whole cell lysates (WCL) and α-GFP immunoprecipitates (IP) from HeLa cells co-transfected with HA-p62 and GFP-SHKBP1 or GFP empty vector (EV) as a control. (B) Western blot analysis of WCL and α-GFP IP from HeLa cells co-transfected with GFP-p62 and HA-SHKBP1 or HA empty vector (EV) as a control. (C and D) Western blot analysis of WCL and α-GFP IP to assess the interaction between SHKBP1 and exogenous (C) or endogenous p62 (D). HeLa cells were transfected with GFP-SHKBP1 in combination with HA-p62 (C) or alone (D), and treated with DMSO or the proteasome inhibitor MG-132 (20 μM) for 2 h or MLN4924 (10 μM) for 16 h. (E) HeLa cells were co-transfected with GFP-p62 and mScarlet-i-SHKBP1, treated with DMSO or the proteasome inhibitor MG-132 (20 μM) for 2 h or MLN4924 (10 μM) for 16 h, and then observed under confocal microscope 24 h post transfection. Scale bars: 10 μm.

    Article Snippet: The following antibodies were used for Western blot: mouse anti-SQSTM1/p62 (D5L7G), Cell Signaling Technology (88588, RRID:AB_2800125); rabbit anti SQSTM1/p62, ABclonal (A19700, RRID:AB_2862742); mouse anti-Keap1, Santa Cruz Biotechnology (sc-365626, RRID:AB_10844829); rabbit anti-SHKBP1, Bethyl Laboratories (A304–891, RRID:AB_2621086); mouse anti-SHKBP1, Santa Cruz Biotechnology (sc-390121, RRID unavailable); mouse anti-ubiquitin, Santa Cruz Biotechnology (sc-8017, RRID:AB_628423); mouse anti-GFP, Santa Cruz Biotechnology (sc-9996, RRID:AB_627695); mouse anti-mCherry, Sigma-Aldrich (MAB131873, RRID:AB_11034849); mouse anti-Actin, MP Bio (08691001, RRID:AB_2335127); rabbit anti-FLAG, Sigma-Aldrich (F7425, RRID:AB_439687); mouse anti-HA, Covance (MMS-101R, RRID:AB_291262); rabbit anti-Tubulin, Cell Signaling Technology (2128S, RRID:AB_823664); rabbit anti-Histone H3, Cell Signaling Technology (4499S, RRID:AB_10544537); mouse anti-Nrf2 (A-10), Santa Cruz Biotechnology (sc-365949, RRID:AB_ 10917561).

    Techniques: Western Blot, Transfection, Plasmid Preparation, Control, Microscopy

    (A) Domain map of SHKBP1 and truncations used in this paper. (B) Representative live-cell images showing localization of full-length (FL) and truncated forms of SHKBP1. HeLa cells were transfected with the indicated GFP-tagged SHKBP1 construct and observed by confocal microscopy 24 h post-transfection. Scale bars: 10 μm. (C) Western blot analysis of α-GFP IP of lysates from HeLa cells co-transfected with HA-CUL3 and either the indicated GFP-SHKBP1 truncation or GFP empty vector (EV) as a control. (D) Western blot analysis of α-GFP IP of lysates from HeLa cells co-transfected with HA-p62 and either the indicated GFP-SHKBP1 truncation or GFP-EV as a control. (E) Domain map of p62 and truncations used in this paper. (F) Representative live cell images showing localization of p62 truncations. HeLa cells were transfected with the indicated GFP-tagged p62 construct and observed by confocal microscopy 24 h post-transfection. Scale bars: 10 μm. (G) Western blot analysis of α-GFP IP of lysates from HeLa cells co-transfected with HA-SHKBP1 and the indicated GFP-p62 construct. (H) AlphaFold3 structural prediction of the interaction between SHKBP1-WD domain (magenta) and p62 (green). Electrostatic interactions (blue) indicated between p62 residues K7, R18, D92, D93 and R96 and SHKBP1 residues R357, K359, D360, D362, and E367 (pink), respectively. ipTM = 0.73, pTM = 0.54. (I) Western blot analysis of α-GFP IP of lysates from HeLa cells co-transfected with the indicated FLAG-SHKBP1 and GFP-p62 constructs. (J) Quantification of FLAG-SHKBP1 from GFP IP samples to assess the strength of interaction between p62 and SHKBP1, with band intensities normalized to FLAG-SHKBP1 levels in WCL and to GFP-p62 levels in IP (n = 3). Exact p values indicated from two-way ANOVA with Tukey’s post-hoc test. (K) Representative live cell images showing localization of p62 point mutants. HeLa cells were transfected with the indicated GFP-tagged p62 construct and observed by confocal microscopy 24 h post transfection. Scale bars: 10 μm.

    Journal: The Journal of cell biology

    Article Title: Cullin-3 adaptor SHKBP1 inhibits SQSTM1/p62 oligomerization and Keap1 sequestration

    doi: 10.1083/jcb.202501207

    Figure Lengend Snippet: (A) Domain map of SHKBP1 and truncations used in this paper. (B) Representative live-cell images showing localization of full-length (FL) and truncated forms of SHKBP1. HeLa cells were transfected with the indicated GFP-tagged SHKBP1 construct and observed by confocal microscopy 24 h post-transfection. Scale bars: 10 μm. (C) Western blot analysis of α-GFP IP of lysates from HeLa cells co-transfected with HA-CUL3 and either the indicated GFP-SHKBP1 truncation or GFP empty vector (EV) as a control. (D) Western blot analysis of α-GFP IP of lysates from HeLa cells co-transfected with HA-p62 and either the indicated GFP-SHKBP1 truncation or GFP-EV as a control. (E) Domain map of p62 and truncations used in this paper. (F) Representative live cell images showing localization of p62 truncations. HeLa cells were transfected with the indicated GFP-tagged p62 construct and observed by confocal microscopy 24 h post-transfection. Scale bars: 10 μm. (G) Western blot analysis of α-GFP IP of lysates from HeLa cells co-transfected with HA-SHKBP1 and the indicated GFP-p62 construct. (H) AlphaFold3 structural prediction of the interaction between SHKBP1-WD domain (magenta) and p62 (green). Electrostatic interactions (blue) indicated between p62 residues K7, R18, D92, D93 and R96 and SHKBP1 residues R357, K359, D360, D362, and E367 (pink), respectively. ipTM = 0.73, pTM = 0.54. (I) Western blot analysis of α-GFP IP of lysates from HeLa cells co-transfected with the indicated FLAG-SHKBP1 and GFP-p62 constructs. (J) Quantification of FLAG-SHKBP1 from GFP IP samples to assess the strength of interaction between p62 and SHKBP1, with band intensities normalized to FLAG-SHKBP1 levels in WCL and to GFP-p62 levels in IP (n = 3). Exact p values indicated from two-way ANOVA with Tukey’s post-hoc test. (K) Representative live cell images showing localization of p62 point mutants. HeLa cells were transfected with the indicated GFP-tagged p62 construct and observed by confocal microscopy 24 h post transfection. Scale bars: 10 μm.

    Article Snippet: The following antibodies were used for Western blot: mouse anti-SQSTM1/p62 (D5L7G), Cell Signaling Technology (88588, RRID:AB_2800125); rabbit anti SQSTM1/p62, ABclonal (A19700, RRID:AB_2862742); mouse anti-Keap1, Santa Cruz Biotechnology (sc-365626, RRID:AB_10844829); rabbit anti-SHKBP1, Bethyl Laboratories (A304–891, RRID:AB_2621086); mouse anti-SHKBP1, Santa Cruz Biotechnology (sc-390121, RRID unavailable); mouse anti-ubiquitin, Santa Cruz Biotechnology (sc-8017, RRID:AB_628423); mouse anti-GFP, Santa Cruz Biotechnology (sc-9996, RRID:AB_627695); mouse anti-mCherry, Sigma-Aldrich (MAB131873, RRID:AB_11034849); mouse anti-Actin, MP Bio (08691001, RRID:AB_2335127); rabbit anti-FLAG, Sigma-Aldrich (F7425, RRID:AB_439687); mouse anti-HA, Covance (MMS-101R, RRID:AB_291262); rabbit anti-Tubulin, Cell Signaling Technology (2128S, RRID:AB_823664); rabbit anti-Histone H3, Cell Signaling Technology (4499S, RRID:AB_10544537); mouse anti-Nrf2 (A-10), Santa Cruz Biotechnology (sc-365949, RRID:AB_ 10917561).

    Techniques: Transfection, Construct, Confocal Microscopy, Western Blot, Plasmid Preparation, Control, Structural Proteomics

    (A) Western blot analysis of in vivo ubiquitination assay. WT or SHKBP1 KO HeLa cells were co-transfected with His-ubiquitin and either GFP-p62 or GFP EV, and overexpressed (OE) group represents WT cells that were also co-transfected with FLAG-SHKBP1. 24 h post transfection, cells were treated with MG-132 (20 μM) for 2 h and subjected to His pull-down using Ni-NTA agarose and Western blot. (B) Western blot analysis of the whole-cell lysates (WCL) from WT, SHKBP1 KO, and FLAG-SHKBP1 overexpressing (OE) HeLa cells. (C) Representative live cell images showing p62 body formation. WT or SHKBP1 KO HeLa cells were transfected with GFP-p62 either alone (left) or in combination with mScarlet-i-SHKBP1 (right, where OE refers to SHKBP1 overexpression in WT cells and RE (rescue) refers to SHKBP1 overexpression in KO cells) and imaged using confocal microscopy 24 h post-transfection. Scale bars: 20 μm. (D) Quantification of the average p62 body size of images shown in (C) from three independently plated samples. n = 51 (KO), 43 (WT), 37 (OE), and 35 (RE). Exact p values indicated (****, p < 0.0001) from one-way ANOVA with Tukey’s post-hoc test. (E) Western blot analysis of WCL from WT or SHKBP1 KO HeLa cells after DSP crosslinking. Cells were treated with MG-132 (0.5 μM) for 12 h, crosslinked with 0.4 mg/mL DSP at 4 °C for 2 h, and lysed in IP lysis buffer with 1% SDS. The lysates mixed with reducing or nonreducing loading buffer (i.e., with or without β-mercaptoethanol) and were analyzed by Western blot. (F) Quantification of the ratio of intensities of monomeric to total p62. Intensities were normalized to the WT without MG-132 treatment group. n = 6. Exact p values indicated (****, p < 0.0001) from two-way ANOVA.

    Journal: The Journal of cell biology

    Article Title: Cullin-3 adaptor SHKBP1 inhibits SQSTM1/p62 oligomerization and Keap1 sequestration

    doi: 10.1083/jcb.202501207

    Figure Lengend Snippet: (A) Western blot analysis of in vivo ubiquitination assay. WT or SHKBP1 KO HeLa cells were co-transfected with His-ubiquitin and either GFP-p62 or GFP EV, and overexpressed (OE) group represents WT cells that were also co-transfected with FLAG-SHKBP1. 24 h post transfection, cells were treated with MG-132 (20 μM) for 2 h and subjected to His pull-down using Ni-NTA agarose and Western blot. (B) Western blot analysis of the whole-cell lysates (WCL) from WT, SHKBP1 KO, and FLAG-SHKBP1 overexpressing (OE) HeLa cells. (C) Representative live cell images showing p62 body formation. WT or SHKBP1 KO HeLa cells were transfected with GFP-p62 either alone (left) or in combination with mScarlet-i-SHKBP1 (right, where OE refers to SHKBP1 overexpression in WT cells and RE (rescue) refers to SHKBP1 overexpression in KO cells) and imaged using confocal microscopy 24 h post-transfection. Scale bars: 20 μm. (D) Quantification of the average p62 body size of images shown in (C) from three independently plated samples. n = 51 (KO), 43 (WT), 37 (OE), and 35 (RE). Exact p values indicated (****, p < 0.0001) from one-way ANOVA with Tukey’s post-hoc test. (E) Western blot analysis of WCL from WT or SHKBP1 KO HeLa cells after DSP crosslinking. Cells were treated with MG-132 (0.5 μM) for 12 h, crosslinked with 0.4 mg/mL DSP at 4 °C for 2 h, and lysed in IP lysis buffer with 1% SDS. The lysates mixed with reducing or nonreducing loading buffer (i.e., with or without β-mercaptoethanol) and were analyzed by Western blot. (F) Quantification of the ratio of intensities of monomeric to total p62. Intensities were normalized to the WT without MG-132 treatment group. n = 6. Exact p values indicated (****, p < 0.0001) from two-way ANOVA.

    Article Snippet: The following antibodies were used for Western blot: mouse anti-SQSTM1/p62 (D5L7G), Cell Signaling Technology (88588, RRID:AB_2800125); rabbit anti SQSTM1/p62, ABclonal (A19700, RRID:AB_2862742); mouse anti-Keap1, Santa Cruz Biotechnology (sc-365626, RRID:AB_10844829); rabbit anti-SHKBP1, Bethyl Laboratories (A304–891, RRID:AB_2621086); mouse anti-SHKBP1, Santa Cruz Biotechnology (sc-390121, RRID unavailable); mouse anti-ubiquitin, Santa Cruz Biotechnology (sc-8017, RRID:AB_628423); mouse anti-GFP, Santa Cruz Biotechnology (sc-9996, RRID:AB_627695); mouse anti-mCherry, Sigma-Aldrich (MAB131873, RRID:AB_11034849); mouse anti-Actin, MP Bio (08691001, RRID:AB_2335127); rabbit anti-FLAG, Sigma-Aldrich (F7425, RRID:AB_439687); mouse anti-HA, Covance (MMS-101R, RRID:AB_291262); rabbit anti-Tubulin, Cell Signaling Technology (2128S, RRID:AB_823664); rabbit anti-Histone H3, Cell Signaling Technology (4499S, RRID:AB_10544537); mouse anti-Nrf2 (A-10), Santa Cruz Biotechnology (sc-365949, RRID:AB_ 10917561).

    Techniques: Ubiquitin Proteomics, Western Blot, In Vivo, Transfection, Over Expression, Confocal Microscopy, Lysis

    (A) WT and SHKBP1 KO HeLa cells were transfected with GFP-p62 alone or in combination with mScarlet-i-SHKBP1. 24 h later, cells were subjected to live-cell imaging by confocal microscopy and analysis by fluorescence recovery after photobleaching (FRAP). Shown is a representative image for each condition. A pre-bleach image is provided along with a timecourse of post-bleach images (post-bleach time indicated in min). Scale bars: 2 μm. (B) Quantification of the fluorescence recovery rates of GFP-p62 from FRAP experiments. Data were analyzed with GraphPad Prism using nonlinear regression (curve fit), shown as mean ± standard deviation (SD). n = 3 for each group from three independent experiments. (C) Quantification of the half-life of fluorescence recovery of GFP-p62 from FRAP experiments. n = 20 for each group from three independent experiments. Exact p values indicated from one-way ANOVA with Tukey’s post-hoc test. (D) Western blot analysis of the whole-cell lysates from three HEK 293T cell lines stably expressing endogenously tagged p62 using mNeonGreen 2 (mNG): SHKBP1 WT, SHKBP1 KO, and SHKBP1 WT with transient overexpression (OE) of a large amount of FLAG-SHKBP1. The hollow arrowhead indicates the FLAG-SHKBP1, and the solid arrowhead indicates untagged (endogenous) SHKBP1. (E) Western blot analysis of WCL, flow-through (FT), and anti-mNeonGreen IP to assess the interaction between SHKBP1 and endogenously tagged p62 in a HEK293T cell line stably expressing mNG-p62 and transfected with a small amount of FLAG-SHKBP1. For the p62 blot, asterisks denote background bands from p62 antibody and solid arrowhead indicates mNG11-tagged p62. For the SHKBP1 blot, hollow arrowhead indicates the FLAG-SHKBP1, and solid arrowhead indicates untagged (endogenous) SHKBP1. (F) Representative live cell images showing single particle tracking of endogenous mNG-p62 by confocal microscopy. HEK 293T cell lines stably expressing mNG-p62 were transfected with either mScarlet-i (in both SHKBP1 KO and SHKBP1 WT backgrounds) or mScarlet-i-SHKBP1 (in SHKBP1 WT background, “OE”). Tracked p62 bodies are indicated by magenta circles, and movement tracks are shown by colored lines. Scale bars: 5 μm. (G) Quantification of the mean speed of tracked p62 bodies in SHKBP1 KO, WT, and OE conditions. n = 200–300 p62 bodies collected from three independently plated samples. Exact p values indicated from one-way ANOVA with Tukey’s post-hoc test. (H) Representative live cell images showing endogenous p62 body shapes. HEK 293T stable cell lines were transfected with the indicated mScarlet-i constructs and observed by confocal microscopy 24 h post-transfection. Scale bars: 5 μm. (I–L) Quantification of the mNG p62 body roundness, solidity, circularity and aspect ratio. n = 1000–1300 p62 bodies collected from three independently plated samples. Exact p values indicated from one-way ANOVA with Tukey’s post-hoc test.

    Journal: The Journal of cell biology

    Article Title: Cullin-3 adaptor SHKBP1 inhibits SQSTM1/p62 oligomerization and Keap1 sequestration

    doi: 10.1083/jcb.202501207

    Figure Lengend Snippet: (A) WT and SHKBP1 KO HeLa cells were transfected with GFP-p62 alone or in combination with mScarlet-i-SHKBP1. 24 h later, cells were subjected to live-cell imaging by confocal microscopy and analysis by fluorescence recovery after photobleaching (FRAP). Shown is a representative image for each condition. A pre-bleach image is provided along with a timecourse of post-bleach images (post-bleach time indicated in min). Scale bars: 2 μm. (B) Quantification of the fluorescence recovery rates of GFP-p62 from FRAP experiments. Data were analyzed with GraphPad Prism using nonlinear regression (curve fit), shown as mean ± standard deviation (SD). n = 3 for each group from three independent experiments. (C) Quantification of the half-life of fluorescence recovery of GFP-p62 from FRAP experiments. n = 20 for each group from three independent experiments. Exact p values indicated from one-way ANOVA with Tukey’s post-hoc test. (D) Western blot analysis of the whole-cell lysates from three HEK 293T cell lines stably expressing endogenously tagged p62 using mNeonGreen 2 (mNG): SHKBP1 WT, SHKBP1 KO, and SHKBP1 WT with transient overexpression (OE) of a large amount of FLAG-SHKBP1. The hollow arrowhead indicates the FLAG-SHKBP1, and the solid arrowhead indicates untagged (endogenous) SHKBP1. (E) Western blot analysis of WCL, flow-through (FT), and anti-mNeonGreen IP to assess the interaction between SHKBP1 and endogenously tagged p62 in a HEK293T cell line stably expressing mNG-p62 and transfected with a small amount of FLAG-SHKBP1. For the p62 blot, asterisks denote background bands from p62 antibody and solid arrowhead indicates mNG11-tagged p62. For the SHKBP1 blot, hollow arrowhead indicates the FLAG-SHKBP1, and solid arrowhead indicates untagged (endogenous) SHKBP1. (F) Representative live cell images showing single particle tracking of endogenous mNG-p62 by confocal microscopy. HEK 293T cell lines stably expressing mNG-p62 were transfected with either mScarlet-i (in both SHKBP1 KO and SHKBP1 WT backgrounds) or mScarlet-i-SHKBP1 (in SHKBP1 WT background, “OE”). Tracked p62 bodies are indicated by magenta circles, and movement tracks are shown by colored lines. Scale bars: 5 μm. (G) Quantification of the mean speed of tracked p62 bodies in SHKBP1 KO, WT, and OE conditions. n = 200–300 p62 bodies collected from three independently plated samples. Exact p values indicated from one-way ANOVA with Tukey’s post-hoc test. (H) Representative live cell images showing endogenous p62 body shapes. HEK 293T stable cell lines were transfected with the indicated mScarlet-i constructs and observed by confocal microscopy 24 h post-transfection. Scale bars: 5 μm. (I–L) Quantification of the mNG p62 body roundness, solidity, circularity and aspect ratio. n = 1000–1300 p62 bodies collected from three independently plated samples. Exact p values indicated from one-way ANOVA with Tukey’s post-hoc test.

    Article Snippet: The following antibodies were used for Western blot: mouse anti-SQSTM1/p62 (D5L7G), Cell Signaling Technology (88588, RRID:AB_2800125); rabbit anti SQSTM1/p62, ABclonal (A19700, RRID:AB_2862742); mouse anti-Keap1, Santa Cruz Biotechnology (sc-365626, RRID:AB_10844829); rabbit anti-SHKBP1, Bethyl Laboratories (A304–891, RRID:AB_2621086); mouse anti-SHKBP1, Santa Cruz Biotechnology (sc-390121, RRID unavailable); mouse anti-ubiquitin, Santa Cruz Biotechnology (sc-8017, RRID:AB_628423); mouse anti-GFP, Santa Cruz Biotechnology (sc-9996, RRID:AB_627695); mouse anti-mCherry, Sigma-Aldrich (MAB131873, RRID:AB_11034849); mouse anti-Actin, MP Bio (08691001, RRID:AB_2335127); rabbit anti-FLAG, Sigma-Aldrich (F7425, RRID:AB_439687); mouse anti-HA, Covance (MMS-101R, RRID:AB_291262); rabbit anti-Tubulin, Cell Signaling Technology (2128S, RRID:AB_823664); rabbit anti-Histone H3, Cell Signaling Technology (4499S, RRID:AB_10544537); mouse anti-Nrf2 (A-10), Santa Cruz Biotechnology (sc-365949, RRID:AB_ 10917561).

    Techniques: Transfection, Live Cell Imaging, Confocal Microscopy, Fluorescence, Standard Deviation, Western Blot, Stable Transfection, Expressing, Over Expression, Single-particle Tracking, Construct

    (A) Western blot analysis of co-IP experiments for endogenous p62. Lysates were from WT HeLa cells, SHKBP1 KO HeLa cells, and HeLa cells transfected with mScarlet-i-SHKBP1 (OE). (B) Quantification of Keap1 from p62-IP to demonstrate the interaction between endogenous p62 and Keap1, with band intensities normalized to actin levels (n = 3). Exact p values indicated from one-way ANOVA with Tukey’s post-hoc test. C) Western blot analysis of WCL from WT and SHKBP1 KO HeLa cells treated with As(III) (10 μM) for the indicated times. (D) Quantification of Keap1 protein levels, with band intensities normalized to actin levels (n = 5). Exact p values indicated (****, p < 0.0001) from two-way ANOVA. (E) Confocal microscopy analysis of WT and KO SHKBP1 HeLa cells co-transfected with GFP-p62 and miRFP-Keap1 and treated with As(III) (10 μM) for 4 h or with DMSO as control. Scale bars: 20 μm. (F and G) Quantification of Keap1 aggregate size (F) and p62 body size (G) in WT and SHKBP1 KO HeLa cells after As(III) treatment (representative images shown in 6E). n = 11 images from three independently plated samples. Exact p values indicated from unpaired two-tailed Student’s t test. (H) Confocal microscopy analysis of HeLa cells co-transfected with GFP-p62 and miRFP-Keap1, with or without mScarlet-i-SHKBP1, and treated with As(III) (10 μM) for 4 h or with DMSO as control. Scale bars: 20 μm. (I and J) Quantification of p62 body size (I) and the percentage of cells containing Keap1 aggregates (J) in control of SHKBP1-overexpressing (OE) HeLa cells with or without As(III) treatment (representative images shown in 6H). n = 8–12 images from three independently plated samples. Exact p values indicated from two-way ANOVA. (K) Immunofluorescence (IF) analysis of endogenous p62 and Keap1 in WT and SHKBP1 KO HeLa cells by confocal microscopy. Cells were treated with As(III) (10 μM) for indicated times. Scale bars: 10 μm; 5 μm (zoomed-in images). (L–N) Quantification of Keap1 aggregate size (L), p62 body size (M), and colocalization of Keap1 and p62 (N) in WT and SHKBP1 KO HeLa cells with As(III) treatment for the indicated times (representative images shown in 6K). n = 13–18 images from three independently plated samples. Exact p values indicated (****, p < 0.0001) from two-way ANOVA with Tukey’s post-hoc test.

    Journal: The Journal of cell biology

    Article Title: Cullin-3 adaptor SHKBP1 inhibits SQSTM1/p62 oligomerization and Keap1 sequestration

    doi: 10.1083/jcb.202501207

    Figure Lengend Snippet: (A) Western blot analysis of co-IP experiments for endogenous p62. Lysates were from WT HeLa cells, SHKBP1 KO HeLa cells, and HeLa cells transfected with mScarlet-i-SHKBP1 (OE). (B) Quantification of Keap1 from p62-IP to demonstrate the interaction between endogenous p62 and Keap1, with band intensities normalized to actin levels (n = 3). Exact p values indicated from one-way ANOVA with Tukey’s post-hoc test. C) Western blot analysis of WCL from WT and SHKBP1 KO HeLa cells treated with As(III) (10 μM) for the indicated times. (D) Quantification of Keap1 protein levels, with band intensities normalized to actin levels (n = 5). Exact p values indicated (****, p < 0.0001) from two-way ANOVA. (E) Confocal microscopy analysis of WT and KO SHKBP1 HeLa cells co-transfected with GFP-p62 and miRFP-Keap1 and treated with As(III) (10 μM) for 4 h or with DMSO as control. Scale bars: 20 μm. (F and G) Quantification of Keap1 aggregate size (F) and p62 body size (G) in WT and SHKBP1 KO HeLa cells after As(III) treatment (representative images shown in 6E). n = 11 images from three independently plated samples. Exact p values indicated from unpaired two-tailed Student’s t test. (H) Confocal microscopy analysis of HeLa cells co-transfected with GFP-p62 and miRFP-Keap1, with or without mScarlet-i-SHKBP1, and treated with As(III) (10 μM) for 4 h or with DMSO as control. Scale bars: 20 μm. (I and J) Quantification of p62 body size (I) and the percentage of cells containing Keap1 aggregates (J) in control of SHKBP1-overexpressing (OE) HeLa cells with or without As(III) treatment (representative images shown in 6H). n = 8–12 images from three independently plated samples. Exact p values indicated from two-way ANOVA. (K) Immunofluorescence (IF) analysis of endogenous p62 and Keap1 in WT and SHKBP1 KO HeLa cells by confocal microscopy. Cells were treated with As(III) (10 μM) for indicated times. Scale bars: 10 μm; 5 μm (zoomed-in images). (L–N) Quantification of Keap1 aggregate size (L), p62 body size (M), and colocalization of Keap1 and p62 (N) in WT and SHKBP1 KO HeLa cells with As(III) treatment for the indicated times (representative images shown in 6K). n = 13–18 images from three independently plated samples. Exact p values indicated (****, p < 0.0001) from two-way ANOVA with Tukey’s post-hoc test.

    Article Snippet: The following antibodies were used for Western blot: mouse anti-SQSTM1/p62 (D5L7G), Cell Signaling Technology (88588, RRID:AB_2800125); rabbit anti SQSTM1/p62, ABclonal (A19700, RRID:AB_2862742); mouse anti-Keap1, Santa Cruz Biotechnology (sc-365626, RRID:AB_10844829); rabbit anti-SHKBP1, Bethyl Laboratories (A304–891, RRID:AB_2621086); mouse anti-SHKBP1, Santa Cruz Biotechnology (sc-390121, RRID unavailable); mouse anti-ubiquitin, Santa Cruz Biotechnology (sc-8017, RRID:AB_628423); mouse anti-GFP, Santa Cruz Biotechnology (sc-9996, RRID:AB_627695); mouse anti-mCherry, Sigma-Aldrich (MAB131873, RRID:AB_11034849); mouse anti-Actin, MP Bio (08691001, RRID:AB_2335127); rabbit anti-FLAG, Sigma-Aldrich (F7425, RRID:AB_439687); mouse anti-HA, Covance (MMS-101R, RRID:AB_291262); rabbit anti-Tubulin, Cell Signaling Technology (2128S, RRID:AB_823664); rabbit anti-Histone H3, Cell Signaling Technology (4499S, RRID:AB_10544537); mouse anti-Nrf2 (A-10), Santa Cruz Biotechnology (sc-365949, RRID:AB_ 10917561).

    Techniques: Western Blot, Co-Immunoprecipitation Assay, Transfection, Confocal Microscopy, Control, Two Tailed Test, Immunofluorescence

    (A) IF analysis of endogenous Nrf2 in WT and SHKBP1 KO HeLa cells by confocal microscopy. Nuclei were stained with DAPI (magenta). Cells were treated with As(III) (10 μM) for the indicated times. Scale bars: 15 μm. (B) Quantification of nuclear levels of Nrf2 in WT and SHKBP1 KO HeLa cells with As(III) treatment for the indicated times (representative images shown in (A). n = 9 images each including ~30 cells from three independently plated samples. Exact p values indicated (****, p < 0.0001) from two-way ANOVA with Tukey’s post-hoc test. (C) Western blot analysis of cytosolic and nuclear fractions from WT and SHKBP1 KO HeLa cells treated with As(III) (10 μM) for the indicated times. Tubulin and Histone H3 are used to qualitatively assess the purity of each fraction. (D) Quantification of nuclear Nrf2 protein levels, with band intensities normalized to Histone H3 levels (n = 3). Exact p values indicated from two-way ANOVA. (E) Role of SHKBP1 in p62-Keap1-Nrf2 pathway.

    Journal: The Journal of cell biology

    Article Title: Cullin-3 adaptor SHKBP1 inhibits SQSTM1/p62 oligomerization and Keap1 sequestration

    doi: 10.1083/jcb.202501207

    Figure Lengend Snippet: (A) IF analysis of endogenous Nrf2 in WT and SHKBP1 KO HeLa cells by confocal microscopy. Nuclei were stained with DAPI (magenta). Cells were treated with As(III) (10 μM) for the indicated times. Scale bars: 15 μm. (B) Quantification of nuclear levels of Nrf2 in WT and SHKBP1 KO HeLa cells with As(III) treatment for the indicated times (representative images shown in (A). n = 9 images each including ~30 cells from three independently plated samples. Exact p values indicated (****, p < 0.0001) from two-way ANOVA with Tukey’s post-hoc test. (C) Western blot analysis of cytosolic and nuclear fractions from WT and SHKBP1 KO HeLa cells treated with As(III) (10 μM) for the indicated times. Tubulin and Histone H3 are used to qualitatively assess the purity of each fraction. (D) Quantification of nuclear Nrf2 protein levels, with band intensities normalized to Histone H3 levels (n = 3). Exact p values indicated from two-way ANOVA. (E) Role of SHKBP1 in p62-Keap1-Nrf2 pathway.

    Article Snippet: The following antibodies were used for Western blot: mouse anti-SQSTM1/p62 (D5L7G), Cell Signaling Technology (88588, RRID:AB_2800125); rabbit anti SQSTM1/p62, ABclonal (A19700, RRID:AB_2862742); mouse anti-Keap1, Santa Cruz Biotechnology (sc-365626, RRID:AB_10844829); rabbit anti-SHKBP1, Bethyl Laboratories (A304–891, RRID:AB_2621086); mouse anti-SHKBP1, Santa Cruz Biotechnology (sc-390121, RRID unavailable); mouse anti-ubiquitin, Santa Cruz Biotechnology (sc-8017, RRID:AB_628423); mouse anti-GFP, Santa Cruz Biotechnology (sc-9996, RRID:AB_627695); mouse anti-mCherry, Sigma-Aldrich (MAB131873, RRID:AB_11034849); mouse anti-Actin, MP Bio (08691001, RRID:AB_2335127); rabbit anti-FLAG, Sigma-Aldrich (F7425, RRID:AB_439687); mouse anti-HA, Covance (MMS-101R, RRID:AB_291262); rabbit anti-Tubulin, Cell Signaling Technology (2128S, RRID:AB_823664); rabbit anti-Histone H3, Cell Signaling Technology (4499S, RRID:AB_10544537); mouse anti-Nrf2 (A-10), Santa Cruz Biotechnology (sc-365949, RRID:AB_ 10917561).

    Techniques: Knock-Out, Translocation Assay, Confocal Microscopy, Staining, Western Blot

    An inducible cell-based model of nuclear, detergent-resistant, p62-positive polyglutamine (polyQ) protein aggregates monitored by an FRET-based reporter. A , schematic of lentiviral constructs for doxycycline-inducible expression of nuclear-localized fluorescent proteins fused to a C-terminal ataxin-3 with 79 glutamines. HEK293T cells engineered with CRISPRi machinery were transduced with these constructs, and clones expressing both fluorescent proteins were selected to generate the NLS-FRET-Q79 cell line. B , fluorescence imaging of NLS-FRET-Q79 cells at 1 day and 5 days of doxycycline. C , immunofluorescence staining for endogenous p62 in NLS-FRET-Q79 cells after 5 days of doxycycline. D , fluorescence imaging of NLS-FRET-Q79 cells at 5 days of doxycycline before and after treatment with detergent, in the same field of view. E , schematic illustrating how polyQ aggregation gives rise to a “FRET-high” population observable by flow cytometry. F , flow cytometry plots at 1 day or 5 days of doxycycline, before and after treatment with detergent. All scale bars represent 10 μm. CRISPRi, CRISPR interference; HEK293T, human embryonic kidney 293T cell line; NLS, nuclear localization signal.

    Journal: The Journal of Biological Chemistry

    Article Title: The Hsp40 cochaperone DNAJC7 regulates polyglutamine aggregation and exhibits context-dependent effects on polyglycine aggregation

    doi: 10.1016/j.jbc.2026.111292

    Figure Lengend Snippet: An inducible cell-based model of nuclear, detergent-resistant, p62-positive polyglutamine (polyQ) protein aggregates monitored by an FRET-based reporter. A , schematic of lentiviral constructs for doxycycline-inducible expression of nuclear-localized fluorescent proteins fused to a C-terminal ataxin-3 with 79 glutamines. HEK293T cells engineered with CRISPRi machinery were transduced with these constructs, and clones expressing both fluorescent proteins were selected to generate the NLS-FRET-Q79 cell line. B , fluorescence imaging of NLS-FRET-Q79 cells at 1 day and 5 days of doxycycline. C , immunofluorescence staining for endogenous p62 in NLS-FRET-Q79 cells after 5 days of doxycycline. D , fluorescence imaging of NLS-FRET-Q79 cells at 5 days of doxycycline before and after treatment with detergent, in the same field of view. E , schematic illustrating how polyQ aggregation gives rise to a “FRET-high” population observable by flow cytometry. F , flow cytometry plots at 1 day or 5 days of doxycycline, before and after treatment with detergent. All scale bars represent 10 μm. CRISPRi, CRISPR interference; HEK293T, human embryonic kidney 293T cell line; NLS, nuclear localization signal.

    Article Snippet: Primary antibody against p62 (clone D5L7G, Cell Signaling, 88588) was diluted 1:1000 in blocking solution and incubated overnight at 4 °C.

    Techniques: Construct, Expressing, Transduction, Clone Assay, Fluorescence, Imaging, Immunofluorescence, Staining, Flow Cytometry, CRISPR

    An inducible model of polyglycine (polyG) aggregation reveals detergent-resistant, p62-positive nuclear inclusions, and seeding activity. A , schematic of lentiviral constructs for generating the NLS-FRET-G100 cell line expressing the upstream ORF (uORF) of the NOTCH2NLC gene with a polyG tract of 100 residues. B , immunofluorescence staining for p62 of NLS-FRET-G100 cells at 5 days of doxycycline. C , fluorescence imaging of NLS-FRET-G100 cells at 5 days of doxycycline before and after detergent treatment, in the same field of view. D , flow cytometry plots at 1 and 5 days of doxycycline treatment and the latter before and after detergent treatment. E , flow cytometry plots of NLS-FRET-G100 cells at 4 days of doxycycline with or without proteasomal inhibitor (carfilzomib). All scale bars represent 10 μm. NLS, nuclear localization signal.

    Journal: The Journal of Biological Chemistry

    Article Title: The Hsp40 cochaperone DNAJC7 regulates polyglutamine aggregation and exhibits context-dependent effects on polyglycine aggregation

    doi: 10.1016/j.jbc.2026.111292

    Figure Lengend Snippet: An inducible model of polyglycine (polyG) aggregation reveals detergent-resistant, p62-positive nuclear inclusions, and seeding activity. A , schematic of lentiviral constructs for generating the NLS-FRET-G100 cell line expressing the upstream ORF (uORF) of the NOTCH2NLC gene with a polyG tract of 100 residues. B , immunofluorescence staining for p62 of NLS-FRET-G100 cells at 5 days of doxycycline. C , fluorescence imaging of NLS-FRET-G100 cells at 5 days of doxycycline before and after detergent treatment, in the same field of view. D , flow cytometry plots at 1 and 5 days of doxycycline treatment and the latter before and after detergent treatment. E , flow cytometry plots of NLS-FRET-G100 cells at 4 days of doxycycline with or without proteasomal inhibitor (carfilzomib). All scale bars represent 10 μm. NLS, nuclear localization signal.

    Article Snippet: Primary antibody against p62 (clone D5L7G, Cell Signaling, 88588) was diluted 1:1000 in blocking solution and incubated overnight at 4 °C.

    Techniques: Activity Assay, Construct, Expressing, Immunofluorescence, Staining, Fluorescence, Imaging, Flow Cytometry

    MAPT p.R406W and isogenic control (WT) neurons at DIV14. A Diagram. B Live imaging using CYTO-ID (green) and microtubule marker (magenta). Rapamycin (Rap, 500 nM); Chloroquine (CQ, 20uM). Scale bar 10 µm. Lower panels, CYTO-ID (green). Scale bar 2 µm. C CYTO-ID mean fluorescence intensity per cell. Cells: WT ( n = 26), WT-Rap+CQ ( n = 29), p.R406W ( n = 34), p.R406W-Rap+CQ ( n = 29). Kruskal–Wallis test followed by Dunn’s test. ** p = 0.0019; **** p < 0.0001. D CYTO-ID density (CYTO-ID+ puncta per cell). Cells: WT ( n = 17), WT-Rap+CQ ( n = 20), p.R406W ( n = 17), p.R406W-Rap+CQ ( n = 19). Kruskal–Wallis test followed by Dunn’s test. * p = 0.0366; * p = 0.0150; ** p = 0.0021; **** p < 0.0001. E Neurons stained for MAP2 (magenta), LC3B (red), and p62 (green). Scale bar 10 µm. F LC3B mean intensity per cell. Cells: MAPT WT ( n = 36) and p.R406W ( n = 47). Two-tailed Mann–Whitney U -test. **** p < 0.0001. G p62 mean intensity per cell. Cells: MAPT WT ( n = 33) and p.R406W ( n = 36). Two-tailed Mann-Whitney U -test. **** p < 0.0001. H Live imaging with microtubule (magenta) and BODIPY (lipid droplets, green). Scale bar 20 µm. Lower panels, BODIPY. Scale bar 2 µm. I BODIPY mean intensity per cell. Cells: MAPT WT ( n = 51) and p.R406W ( n = 61). Mann–Whitney U -test. **** p < 0.0001. J Schematic. K Neurons expressing the pHluorin-mKate2-LC3 reporter. Scale bar 5 μm. Right panel: puncta/autophagic vesicles, pHLuorin (green), mKate2 (red), merged signals (yellow). Scale bars 1 µm. L Puncta size. Puncta: WT ( n = 48), p.R406W ( n = 87), WT-Rap+CQ ( n = 181). Kruskal-Wallis test followed by Dunn’s test. *** p = 0.0002; **** p < 0.0001. M mKate2 to pHLuorin ratio. Puncta: WT ( n = 64), p.R406W ( n = 87), WT-Rap+CQ ( n = 181). Kruskal–Wallis test followed by Dunn’s test. *** p = 0.0005, **** p < 0.0001. N Number of autophagosomes (mKate + / pHLuorin + ) and autolysosomes (mKate + /pHLuorin-) puncta. Total puncta: MAPT WT ( n = 64), p.R406W ( n = 87); WT-Rap+CQ ( n = 111). Autolysosomes: MAPT WT ( n = 53), p.R406W ( n = 27), WT-Rap+CQ ( n = 15). Autophagosomes: MAPT WT ( n = 11), p.R406W ( n = 60), WT-Rap+CQ ( n = 70). Two-way ANOVA followed by Tukey multiple comparisons test, * p = 0.0205; * p = 0.0146; ** p = 0.0059; *** p = 0.0006; *** p = 0.0008. WT ( n = 12 cells), MAPT p.R406W ( n = 11 cells), WT-Rap+CQ ( n = 13 cells). All data are represented as mean ± SEM from 3 independent experiments. Source data are provided as a Source Data file. Created in BioRender. Mirfakhar, F. (2026) https://BioRender.com/8bzn5l2 .

    Journal: Nature Communications

    Article Title: A pathogenic Tau mutation drives autophagy-lysosome dysfunction that limits Tau degradation in a model of frontotemporal dementia

    doi: 10.1038/s41467-026-70473-5

    Figure Lengend Snippet: MAPT p.R406W and isogenic control (WT) neurons at DIV14. A Diagram. B Live imaging using CYTO-ID (green) and microtubule marker (magenta). Rapamycin (Rap, 500 nM); Chloroquine (CQ, 20uM). Scale bar 10 µm. Lower panels, CYTO-ID (green). Scale bar 2 µm. C CYTO-ID mean fluorescence intensity per cell. Cells: WT ( n = 26), WT-Rap+CQ ( n = 29), p.R406W ( n = 34), p.R406W-Rap+CQ ( n = 29). Kruskal–Wallis test followed by Dunn’s test. ** p = 0.0019; **** p < 0.0001. D CYTO-ID density (CYTO-ID+ puncta per cell). Cells: WT ( n = 17), WT-Rap+CQ ( n = 20), p.R406W ( n = 17), p.R406W-Rap+CQ ( n = 19). Kruskal–Wallis test followed by Dunn’s test. * p = 0.0366; * p = 0.0150; ** p = 0.0021; **** p < 0.0001. E Neurons stained for MAP2 (magenta), LC3B (red), and p62 (green). Scale bar 10 µm. F LC3B mean intensity per cell. Cells: MAPT WT ( n = 36) and p.R406W ( n = 47). Two-tailed Mann–Whitney U -test. **** p < 0.0001. G p62 mean intensity per cell. Cells: MAPT WT ( n = 33) and p.R406W ( n = 36). Two-tailed Mann-Whitney U -test. **** p < 0.0001. H Live imaging with microtubule (magenta) and BODIPY (lipid droplets, green). Scale bar 20 µm. Lower panels, BODIPY. Scale bar 2 µm. I BODIPY mean intensity per cell. Cells: MAPT WT ( n = 51) and p.R406W ( n = 61). Mann–Whitney U -test. **** p < 0.0001. J Schematic. K Neurons expressing the pHluorin-mKate2-LC3 reporter. Scale bar 5 μm. Right panel: puncta/autophagic vesicles, pHLuorin (green), mKate2 (red), merged signals (yellow). Scale bars 1 µm. L Puncta size. Puncta: WT ( n = 48), p.R406W ( n = 87), WT-Rap+CQ ( n = 181). Kruskal-Wallis test followed by Dunn’s test. *** p = 0.0002; **** p < 0.0001. M mKate2 to pHLuorin ratio. Puncta: WT ( n = 64), p.R406W ( n = 87), WT-Rap+CQ ( n = 181). Kruskal–Wallis test followed by Dunn’s test. *** p = 0.0005, **** p < 0.0001. N Number of autophagosomes (mKate + / pHLuorin + ) and autolysosomes (mKate + /pHLuorin-) puncta. Total puncta: MAPT WT ( n = 64), p.R406W ( n = 87); WT-Rap+CQ ( n = 111). Autolysosomes: MAPT WT ( n = 53), p.R406W ( n = 27), WT-Rap+CQ ( n = 15). Autophagosomes: MAPT WT ( n = 11), p.R406W ( n = 60), WT-Rap+CQ ( n = 70). Two-way ANOVA followed by Tukey multiple comparisons test, * p = 0.0205; * p = 0.0146; ** p = 0.0059; *** p = 0.0006; *** p = 0.0008. WT ( n = 12 cells), MAPT p.R406W ( n = 11 cells), WT-Rap+CQ ( n = 13 cells). All data are represented as mean ± SEM from 3 independent experiments. Source data are provided as a Source Data file. Created in BioRender. Mirfakhar, F. (2026) https://BioRender.com/8bzn5l2 .

    Article Snippet: Primary antibodies used for immunostaining included: anti-MAP2 (Abcam, ab5392), anti-LAMP1 (D2D11; Cell signaling, 9091), anti-LAMP1 (D4O1S; Cell signaling, 15665), anti-Tau5 (total Tau; generously provided by Dr. Lester Binder), anti-AT180 (pTau-Thr23; Thermo Fisher Scientific, MN1040), anti-AT8 (pTau-Ser202, Thr 205; Thermo Scientific, MN1020B), anti-JIP3 (Thermo Fisher, PA5-59728), anti-LC3B (Cell Signaling, 2775), anti-SQSTM1 / p62 (Cell signaling, D5L7G 88588S), anti-Synapsin I (Millipore, AB1543), anti-Cathepsin D (C-5; SantaCruz, sc-377124), anti-Limp2 (Thermo Fisher Scientific, 712072), anti-Beta III Tubulin (Tuj1; Promega, G712A), and DAPI (Sigma, D-9542).

    Techniques: Control, Imaging, Marker, Fluorescence, Staining, Two Tailed Test, MANN-WHITNEY, Expressing

    MAPT p.R406W and isogenic control (WT) neurons were treated with G2-567 (0.5 µM) or DMSO for 14 days beginning on DIV7 and fixed on DIV21. Neurons were then immunostained for lysosomal positioning and autophagy markers. A Immunostaining for MAP2 (green) and LAMP1 (red). Lower panel, 3D reconstruction by Imaris. Scale bar 5 µm. B Quantification of lysosome density: WT ( n = 16), WT-G2-567 ( n = 14), p.R406W ( n = 17), p.R406W-G2-567 ( n = 19). Kruskal-Wallis test followed by Dunn’s test. * p = 0.0214, p = 0.0390; ** p = 0.0017. C Quantification of lysosome distance from the nucleus as the shortest distance of LAMP1+ vesicles from DAPI. Quantification performed by Imaris 3D rendering. Data on the y -axis are normalized to WT-DMSO. Number of vesicles quantified: WT ( n = 181), WT-G2-567 ( n = 219), p.R406W ( n = 319), p.R406W-G2-567 ( n = 318). Kruskal–Wallis test followed by Dunn’s test. WT-DMSO vs. p.R406W-DMSO: ** p = 0.0011. p.R406W-DMSO vs. p.R406W-G2-567: p = 0.3235. D Immunostaining with MAP2+ (magenta) and JIP3 (green). Scale bar 10 µm. E Quantification of JIP3 mean intensity per cell. WT (n = 43), WT-G2-567 ( n = 55), p.R406W ( n = 33), p.R406W-G2-567 ( n = 54) cells. Kruskal–Wallis test followed by Dunn’s test; **** p < 0.0001. F Cells were probed for autophagosomes using CYTO-ID (green) and microtubule (magenta). Right panel, magnified CYTO-ID positive vesicles (green). Scale bar 5 µm. G Quantification of CYTO-ID mean density. Total number of cells quantified: WT ( n = 71), WT-G2-567 ( n = 79), p.R406W ( n = 79), p.R406W-G2-567 ( n = 78). Kruskal–Wallis test followed by Dunn’s test, ** p = 0.0027; **** p < 0.0001. H Immunostaining for MAP2 (magenta), LC3B (red) and p62 (green). Scale bar 10 µm. I Quantification of LC3B mean intensity per cell. WT ( n = 70), WT-G2-567 ( n = 43), p.R406W ( n = 68), p.R406W-G2-567 ( n = 68) cells. Kruskal-Wallis test followed by Dunn’s test, * p = 0.0393; ** p = 0.0062; *** p = 0.0005; **** p < 0.0001. J Quantification of p62 mean intensity. WT ( n = 26), WT-G2-567 ( n = 32), p.R406W ( n = 32), p.R406W-G2-567 ( n = 26) cells. Kruskal–Wallis test. * p = 0.0266, **** p < 0.0001. All data are mean ± SEM. All data are normalized to WT-DMSO. Data are representative of 3 independent experiments except in panels I and J which represent 4 independent experiments. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: A pathogenic Tau mutation drives autophagy-lysosome dysfunction that limits Tau degradation in a model of frontotemporal dementia

    doi: 10.1038/s41467-026-70473-5

    Figure Lengend Snippet: MAPT p.R406W and isogenic control (WT) neurons were treated with G2-567 (0.5 µM) or DMSO for 14 days beginning on DIV7 and fixed on DIV21. Neurons were then immunostained for lysosomal positioning and autophagy markers. A Immunostaining for MAP2 (green) and LAMP1 (red). Lower panel, 3D reconstruction by Imaris. Scale bar 5 µm. B Quantification of lysosome density: WT ( n = 16), WT-G2-567 ( n = 14), p.R406W ( n = 17), p.R406W-G2-567 ( n = 19). Kruskal-Wallis test followed by Dunn’s test. * p = 0.0214, p = 0.0390; ** p = 0.0017. C Quantification of lysosome distance from the nucleus as the shortest distance of LAMP1+ vesicles from DAPI. Quantification performed by Imaris 3D rendering. Data on the y -axis are normalized to WT-DMSO. Number of vesicles quantified: WT ( n = 181), WT-G2-567 ( n = 219), p.R406W ( n = 319), p.R406W-G2-567 ( n = 318). Kruskal–Wallis test followed by Dunn’s test. WT-DMSO vs. p.R406W-DMSO: ** p = 0.0011. p.R406W-DMSO vs. p.R406W-G2-567: p = 0.3235. D Immunostaining with MAP2+ (magenta) and JIP3 (green). Scale bar 10 µm. E Quantification of JIP3 mean intensity per cell. WT (n = 43), WT-G2-567 ( n = 55), p.R406W ( n = 33), p.R406W-G2-567 ( n = 54) cells. Kruskal–Wallis test followed by Dunn’s test; **** p < 0.0001. F Cells were probed for autophagosomes using CYTO-ID (green) and microtubule (magenta). Right panel, magnified CYTO-ID positive vesicles (green). Scale bar 5 µm. G Quantification of CYTO-ID mean density. Total number of cells quantified: WT ( n = 71), WT-G2-567 ( n = 79), p.R406W ( n = 79), p.R406W-G2-567 ( n = 78). Kruskal–Wallis test followed by Dunn’s test, ** p = 0.0027; **** p < 0.0001. H Immunostaining for MAP2 (magenta), LC3B (red) and p62 (green). Scale bar 10 µm. I Quantification of LC3B mean intensity per cell. WT ( n = 70), WT-G2-567 ( n = 43), p.R406W ( n = 68), p.R406W-G2-567 ( n = 68) cells. Kruskal-Wallis test followed by Dunn’s test, * p = 0.0393; ** p = 0.0062; *** p = 0.0005; **** p < 0.0001. J Quantification of p62 mean intensity. WT ( n = 26), WT-G2-567 ( n = 32), p.R406W ( n = 32), p.R406W-G2-567 ( n = 26) cells. Kruskal–Wallis test. * p = 0.0266, **** p < 0.0001. All data are mean ± SEM. All data are normalized to WT-DMSO. Data are representative of 3 independent experiments except in panels I and J which represent 4 independent experiments. Source data are provided as a Source Data file.

    Article Snippet: Primary antibodies used for immunostaining included: anti-MAP2 (Abcam, ab5392), anti-LAMP1 (D2D11; Cell signaling, 9091), anti-LAMP1 (D4O1S; Cell signaling, 15665), anti-Tau5 (total Tau; generously provided by Dr. Lester Binder), anti-AT180 (pTau-Thr23; Thermo Fisher Scientific, MN1040), anti-AT8 (pTau-Ser202, Thr 205; Thermo Scientific, MN1020B), anti-JIP3 (Thermo Fisher, PA5-59728), anti-LC3B (Cell Signaling, 2775), anti-SQSTM1 / p62 (Cell signaling, D5L7G 88588S), anti-Synapsin I (Millipore, AB1543), anti-Cathepsin D (C-5; SantaCruz, sc-377124), anti-Limp2 (Thermo Fisher Scientific, 712072), anti-Beta III Tubulin (Tuj1; Promega, G712A), and DAPI (Sigma, D-9542).

    Techniques: Control, Immunostaining