rabbit polyclonal anti phospho p62 s403  (Cell Signaling Technology Inc)

Journal: Molecules and Cells

Article Title: CpG oligodeoxynucleotide reduces PrP Sc accumulation and prolongs survival in prion-infected mice

doi: 10.1016/j.mocell.2026.100335

Figure Lengend Snippet: CpG ODN induces AMPK signaling and attenuates phosphorylated p62 accumulation in 22L scrapie-infected mice. (a) Western blot analysis of brain lysates at 170 dpi showing expression levels of p-AMPK T172, AMPK, p-ULK1 S555, ULK1, p-p62 S403, p62, ATG12–5, and LC3 I/II in brains of 22L scrapie-infected mice with or without CpG ODN at 170 dpi. (b) Relative intensity of p-AMPK, p-ULK, p62, p-p62, ATG12–5, and LC3-II represented as bar graphs (mean ± S.E.M, n = 6). Statistical significance was determined by 1-way ANOVA with Tukey’s post hoc test. * P < .05, ** P < .01, *** P < .001. NS: not significant.

Article Snippet: Membranes were blocked with 5% nonfat dry milk in PBST (8 mM Na 2 HPO 4 , 2 mM KH 2 PO4, 138 mM NaCl, 2.7 mM KCl, 0.1% Tween 20; pH 7.4) for 1 hour at room temperature (RT), followed by overnight incubation at 4 °C with the following primary antibodies: mouse monoclonal anti-PrP 3F10 (1:2000) , rabbit polyclonal anti-TLR9 (1:2000, Abcam, Cambridge, UK), rabbit polyclonal anti-phospho AMPK T172 (p-AMPK T172) (1:2000, Cell Signaling Technology, Danvers, MA, USA), rabbit monoclonal anti-AMPK (1:2000, Cell Signaling Technology), rabbit polyclonal anti-phospho-p62 S403 (p-p62 S403) (1:2000, Cell Signaling Technology), rabbit monoclonal anti-p62 (1:2000, MBL, Nagoya, Japan), rabbit monoclonal anti-phospho-ULK1 S555 (p-ULK1 S555)(1:2000, Cell Signaling Technology), rabbit polyclonal anti-ULK1 (1:2000, Cell Signaling Technology), rabbit polyclonal anti-LC3 I/II (1:2000, Cell Signaling Technology), rabbit polyclonal anti-GFAP (1:2000, CosmoBio, Tokyo, Japan), rabbit polyclonal anti-ATG12 (1:2000, Cell Signaling Technology) and mouse monoclonal anti-β-actin (1:2000, Sigma-Aldrich).

Techniques: Infection, Western Blot, Expressing

Journal: Molecules and Cells

Article Title: CpG oligodeoxynucleotide reduces PrP Sc accumulation and prolongs survival in prion-infected mice

doi: 10.1016/j.mocell.2026.100335

Figure Lengend Snippet: CpG ODN reduces PrP Sc accumulation and activates AMPK-associated signaling in 22L scrapie-infected neuronal cells. (a and b) Immunoblot analysis of (a) PrP Sc and total PrP, and (b) TLR9, p-AMPK T172, total AMPK, p-ULK1 S555, total ULK1, ATG12–5, total p62, and LC3 I/II in 22L scrapie-infected neuronal cells (ZW-22L) treated with CpG ODN (0, 1, or 3 µM) for 6 hours. For PrP Sc detection, equal amounts of protein were incubated with proteinase K (2 µg/ml) for 1 hour. (c) Densitometric quantification of p-AMPK, p-ULK1, ATG12–5, total p62, and LC3-II levels. Data are presented as mean ± S.E.M ( n = 3). (d) Immunoblot analysis of PrP Sc , p-AMPK T172, and total AMPK in ZW-22L cells treated with CpG ODN (3 µM, 6 hours) in presence or absence of the TLR9 antagonist ODN 2088 (5 µM, 7 hours). Data represents 3 independent experiments ( n = 3). Statistical significance was determined by 1-way ANOVA with Tukey’s post hoc test (* P < .05, *** P < .001).

Article Snippet: Membranes were blocked with 5% nonfat dry milk in PBST (8 mM Na 2 HPO 4 , 2 mM KH 2 PO4, 138 mM NaCl, 2.7 mM KCl, 0.1% Tween 20; pH 7.4) for 1 hour at room temperature (RT), followed by overnight incubation at 4 °C with the following primary antibodies: mouse monoclonal anti-PrP 3F10 (1:2000) , rabbit polyclonal anti-TLR9 (1:2000, Abcam, Cambridge, UK), rabbit polyclonal anti-phospho AMPK T172 (p-AMPK T172) (1:2000, Cell Signaling Technology, Danvers, MA, USA), rabbit monoclonal anti-AMPK (1:2000, Cell Signaling Technology), rabbit polyclonal anti-phospho-p62 S403 (p-p62 S403) (1:2000, Cell Signaling Technology), rabbit monoclonal anti-p62 (1:2000, MBL, Nagoya, Japan), rabbit monoclonal anti-phospho-ULK1 S555 (p-ULK1 S555)(1:2000, Cell Signaling Technology), rabbit polyclonal anti-ULK1 (1:2000, Cell Signaling Technology), rabbit polyclonal anti-LC3 I/II (1:2000, Cell Signaling Technology), rabbit polyclonal anti-GFAP (1:2000, CosmoBio, Tokyo, Japan), rabbit polyclonal anti-ATG12 (1:2000, Cell Signaling Technology) and mouse monoclonal anti-β-actin (1:2000, Sigma-Aldrich).

Techniques: Infection, Western Blot, Incubation

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: Sections were then incubated overnight at 4 °C with a rodent-specific anti-p62 antibody (clone D6M5X, Cell Signaling, 23214) at a 1:500 dilution in blocking buffer.

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

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: Sections were then incubated overnight at 4 °C with a rodent-specific anti-p62 antibody (clone D6M5X, Cell Signaling, 23214) at a 1:500 dilution in blocking buffer.

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

Journal: Redox Biology

Article Title: YY1 nitration participates in DbCM cardiomyocyte lipotoxicity by inhibiting ANXA3 -induced microlipophagy

doi: 10.1016/j.redox.2026.104085

Figure Lengend Snippet: ANXA3 did not regulate myocardial free fatty acid uptake or lipolysis but positively modulated macroautophagy in T2DM mice. (A-C) Relative p62 protein expression level and LC3 II/I ratio in heart tissues of db/m and db/db mice analyzed by Western blot, n = 4. (D-F) Relative p62 protein expression level and LC3 II/I ratio in heart tissues of mice analyzed by Western blot, n = 4. (G-I) Relative p62 protein expression level and LC3 II/I ratio in AC16 cardiomyocytes analyzed by Western blot, n = 6. (J-L) Relative p62 protein expression level and LC3 II/I ratio in AC16 cardiomyocytes analyzed by Western blot, n = 6-8. The data were presented as the mean ± SD. ∗ P < 0.05 versus the db/m group or the db/db + OE-GFP group; ∗∗ P < 0.01 versus the db/m group or the vehicle group or the OE-NC + vehicle group; ∗∗∗ P < 0.001 versus the vehicle group or the OE-NC + vehicle group; # P < 0.05 versus the OE-NC + HGPA group; ### P < 0.001 versus the OE-NC + HGPA group.

Article Snippet: Blocked with 5% (w/v) non-fat-dried milkat room temperature for 1 h. Then the membranes were incubated with the anti-ANXA3 antibody (Proteintech, 11804-1-AP; 1:1000 [v/v]), the anti-YY1 antibody (Proteintech, 22156-1-AP; 1:1000 [v/v]) the anti-PLIN2 antibody (Proteintech, 15294-1-AP; 1:1000 [v/v]), the anti-SQSTM1/p62 antibody (Cell Signaling Technology, 23214; 1:1000 [v/v]), the anti-LC3 antibody (Cell Signaling Technology, 12741; 1:1000 [v/v]), the anti-Rab7 antibody (Proteintech, 55469-1-AP; 1:1000 [v/v])or the rabbit anti-α-Tubulin antibody (ABclonal, AC031; 1:1000 [v/v])overnight at 4 °C.

Techniques: Expressing, Western Blot