rabbit polyclonal anti p62/sqstm1  (Proteintech)

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: Cell Death Discovery

Article Title: Inhibiting HSP27 activates the XBP1s/CerS1 interplay, which triggers DRP1-driven mitophagy, thereby protecting against cell death and promoting the KSHV lytic cycle in primary effusion lymphoma cells

doi: 10.1038/s41420-026-02979-2

Figure Lengend Snippet: 4μ8 C ( F ) or P053 ( G ) or Mdivi-1 (DRP1 inhibitor) ( H ). Mitophagy was evaluated following treatment with J2 with or without Mdivi-1 (DRP1inhibitor) ( A ). Cells treated with CCCP (Mitophagy inducer) were used as positive control. BC3 cells were stained with MitoTracker (a mitochondrial marker, green) and Lysotracker (a lysosomal marker, red). Yellow puncta resulting from the co-localization of MitoTracker (a mitochondrial marker green) and LysoTracker (a lysosomal marker red) were evaluated in control and J2-treated cells. The histograms indicate yellow puncta/cell. The data are represented as the mean plus S.D. of three different experiments. p value * <0.05, as calculated by ANOVA test. Scale bar = 10 µm. B Western blot analysis of p62 and LC3I/II in BC3 cells cultured with Mdivi-1 (DRP1 inhibitor) and subsequently treated with J2 or with J2 alone. The histograms represent the densitometric analysis of the ratio of p62/GAPDH and LC3II/GAPDH. The data are represented as the mean plus S.D. of three different experiments. p value * <0.05, as calculated by ANOVA test. C Expression of HADHA as evaluated by western blot analysis in PEL cell lines pretreated with Mdivi-1 (DRP1 inhibitor) before J2 treatment (20 μM). Histone H3 and GAPDH were used as loading controls. The histograms represent the densitometric analysis of the ratio of HADHA /H3 and HADHA /GAPDH. The data are represented as the mean plus S.D. of three different experiments. p value * <0.05, as calculated by ANOVA test. D BC3 and BCBL1 cell lines were knocked down for DRP1 (siDRP1) or scramble-treated for 24 h, exposed or not to J2 and finally evaluated for the expression of DRP1 and HADHA by western blot analysis. The histograms represent the densitometric analysis of the ratio of DRP1/GAPDH and HADHA/GAPDH. The data are represented as the mean plus S.D. of three different experiments. p value * <0.05, as calculated by ANOVA test. E Western blot analysis of HADHA in BC3 cells cultured with 4μ8 C (Ire1α endoribonuclease activity inhibitor) and subsequently treated with J2 or with J2 alone for 4 h and ( F ) in BC3 cells cultured with P053 (CerS1 inhibitor) and then treated with J2 or with J2 alone for 4 h. The histograms represent the densitometric analysis of the ratio HADHA/GAPDH. The data are represented as the mean plus S.D. of three different experiments. p value * <0.05, as calculated by ANOVA test.

Article Snippet: The following primary antibodies were used in western blots: rabbit polyclonal anti-PARP (1:1000) (Cell Signaling, Danvers, MA, USA, cat n. 9542), mouse monoclonal anti-CerS1 (LASS1) (1:500) (Santa Cruz Biotechnology Inc., Dallas, TX, USA, cat. n. 293497), rabbit polyclonal anti-BiP/GRIP78 (1:5000) (; Proteintech, Rosemont, IL, USA, cat n. 11587-1-AP), rabbit polyclonal anti-CHOP (GADD153) (1:1000) (Proteintech, Rosemont, IL, USA, cat n. 15204-1-AP), rabbit polyclonal anti-XBP1 (1:500) (Novus Biologicals, Centennial, CO, USA; cat n. NBP1-77681SS), mouse monoclonal anti-DRP1 (1:500) (Santa Cruz Biotechnology Inc., Dallas, TX, USA, cat. n. sc-271583), mouse monoclonal anti-HADHA (1:500) (Santa Cruz Biotechnology Inc., Dallas, TX, USA, cat. n. sc-374497), mouse monoclonal anti-Parkin (1:500) (Santa Cruz Biotechnology Inc., Dallas, TX, USA, cat. n. sc-32283), rabbit polyclonal anti-LC3 I/II (1:1000) (Novus Biologicals, Centennial, CO, USA cat. n. NB100–2220), rabbit polyclonal anti-SQSTM1/p62 (1:500) (Novus Biologicals, Centennial, CO, USA cat. n. NBP-48-320), mouse monoclonal anti-HSP27 (1:1000) (Proteintech, Rosemont, IL, USA, cat n. 182841), rabbit polyclonal anti-HSP110 (1:200) (Abcam, Cambridge UK, cat. n. ab24503).

Techniques: Positive Control, Staining, Marker, Control, Western Blot, Cell Culture, Expressing, Activity Assay

Journal: Journal of Neuroinflammation

Article Title: Selective vulnerability of the aging cholinergic system to amyloid pathology revealed by induced APP overexpression

doi: 10.1186/s12974-025-03682-2

Figure Lengend Snippet: Autophagic activity in the mouse brain was modulated by both aging and mutant APP transgene expression. a Representative western blot images are shown. Autophagy-related targets assessed include ( b ) LC3A-II/LC3A-I ratio, ( c ) total LC3A, ( d ) LC3B-II/LC3B-I ratio, ( e ) total LC3B, ( f ) ATG3, ( g ) ATG5, ( h ) ATG7, ( i ) ATG12, ( j ) Beclin-1, and ( k ) SQSTM1/p62. Sample size was 5 male and 5 female 18mo controls, 4–5 male and 6 female APP 6 → 18mo, 4–5 male and 5 female 24mo controls, and 5 male and 5–6 female APP 12 → 24mo. Individual data points and group means ± S.E.M. are presented. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: The following primary antibodies were used: mouse monoclonal anti-human Aβ (#SIG-39300, clone 6E10, 1:2,000; Covance, Princeton, NJ, USA), rabbit monoclonal anti-BACE1 (#5606, clone D10E5, 1:2,000; Cell Signaling Technology, Danvers, MA, USA), rabbit monoclonal anti-presenilin 1 (#5643, clone D39D1, 1:3,000; Cell Signaling Technology), rabbit monoclonal anti-LC3A (#4599, clone D50G8, 1:2,000; Cell Signaling Technology), rabbit polyclonal anti-LC3B (#2775, 1:2,000; Cell Signaling Technology), rabbit polyclonal anti-ATG3 (#3415, 1:2,000; Cell Signaling Technology), rabbit monoclonal anti-ATG5 (#8540, clone D1G9, 1:2,000; Cell Signaling Technology), rabbit monoclonal anti-ATG7 (#8558, clone D12B11, 1:2,000; Cell Signaling Technology), rabbit monoclonal anti-ATG12 (#4180, clone D88H11, 1:2,000; Cell Signaling Technology), rabbit monoclonal anti-beclin 1 (#3495, clone D40C5, 1:2,000; Cell Signaling Technology), rabbit polyclonal anti-p62 (#5114, 1:1,500; Cell Signaling Technology), rabbit monoclonal anti-ChAT (#ab181023, clone EPR13024 (B), 1:2,000; Abcam, Cambridge, UK), rabbit polyclonal anti-SLC5A7 (#ab135043, 1:3,000; Abcam), rabbit polyclonal anti-PSD95 (#2507, 1:2,000; Cell Signaling Technology), mouse monoclonal anti-synaptophysin (#ab8049, clone SY38, 1:2,000; Abcam), rabbit anti GFAP (#12389, clone D1F4Q, 1:2,000; Cell Signaling Technology), rabbit anti CD11b (#17800, clone E6E1M, 1:2,000; Cell Signaling Technology), rabbit anti CD68 (#97778, clone E3O7V, 1:2,000; Cell Signaling Technology), rabbit anti Iba1/AIF-1 (#17198, clone E4O4W, 1:2,000; Cell Signaling Technology), rabbit anti TREM2 (#59621, clone E9O9F, 1:2,000; Cell Signaling Technology), mouse monoclonal anti-actin (#869100, clone C4, 1:20,000; MP Biomedicals, Santa Ana, CA, USA).

Techniques: Activity Assay, Mutagenesis, Expressing, Western Blot