mouse monoclonal anti sqstm1  (Novus Biologicals)

Journal: bioRxiv

Article Title: VPS13C/PARK23 initiates lipid transfer and membrane remodeling for efficient lysosomal repair

doi: 10.1101/2025.10.23.684214

Figure Lengend Snippet: ( a ) Volcano plot showing enrichment of VPS13C, OSBP and various lysosomal damage markers on lysosomes affinity-purified from HeLa cells treated with LLOMe (500 μM, 30 min). Green , proteins enriched in both intact (–LLOMe) and damaged (+LLOMe) lysosomes; blue , proteins selectively enriched in intact lysosomes; magenta , proteins selectively enriched in damaged lysosomes. ( b ) Whole cell lysates (WC) and lysosomes purified from untreated and LLOMe-treated HeLa cells (500 μM, 30 min) were subjected to immunoblot analysis using antibodies against VPS13C, OSBP, LAMP1, PI4K2A, hIST1, Gal3/LEG3 and p62/SQSTM. ( c ) Quantification of the data in (b). Bars show normalized values relative to control. Data are means ± SD. n = 4 biological replicates. ( d ) Time-lapse fluorescence images of U2OS wildtype (WT) and PI4K2A-KO cells expressing VPS13C-mClover or OSBP-GFP ( green ) that were fed CF633-labelled 70kDa dextran ( magenta ) and treated with 1 mM LLOMe for the indicated time. Cells were imaged by spinning disk microscopy. Scale bar, 5 µm. ( e ) Time-course plotting VPS13C- and OSBP-positive puncta per 100 μm area in cells treated as in (d). Data are means ± SD (VPS13C: n = 31 cells for WT, 29 cells for PI4K2A-KO; OSBP: n = 35 cells for WT, 33 cells for PI4K2A-KO) from 2 independent experiments.

Article Snippet: Antibodies used were: rabbit polyclonal anti-hIST1 (Proteintech, 19842; IB: 1:1000; IF 1:1000), rabbit polyclonal anti-OSBP (Proteintech, 11096-1-AP; IB: 1:500; IF: 1:200), mouse monoclonal anti-PI4-kinase II Alpha (B5) (Santa Cruz, sc-390026; IB 1:1000; IF 1:400), rabbit polyclonal anti-VPS13C (Proteintech, 28676-1-AP; IB 1:1000), mouse monoclonal anti-α-Actin (Sigma, A1978; IB 1:50,000), mouse monoclonal anti-Mitochondrial surface p60 (Millipore, MAB1273; IB 1:1000), mouse monoclonal anti-Na + /K + ATPase (Santa Cruz, sc-48345; IB 1:1000), mouse monoclonal anti-LAMP1 (H4A3) (Santa Cruz, sc-20011; IB 1:1000; IF 1:400), rabbit polyclonal anti-Calnexin (Santa Cruz, sc-11397; IB 1:1000), mouse monoclonal anti-Galectin 3 (B2C10) (Santa Cruz, sc-32790; IB 1:1000), mouse monoclonal anti-VAPA (4C12) (Santa Cruz, sc-293278; IF 1:400), mouse monoclonal anti-p62/SQSTM (D-3) (Santa Cruz, sc-28359; IB 1:500), mouse monoclonal anti-Alix (BioLegend, 634502; IF 1:200), mouse monoclonal anti-ORP-9 (A7) (Santa Cruz, sc-398961; IF 1:200), HRP-conjugated goat anti-mouse IgG (Thermo Fisher Scientific, 31430; IB 1:5000), HRP-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific; 31460; IB 1:5000), Cyanine CyTM2-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories, 715-225-150; IF 1:250), Cyanine CyTM2-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, 715-225-152; IF 1:250), Cyanine CyTM3-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories, 715-162-150; IF 1:250), Cyanine CyTM3-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, 711-165-152; IF 1:250), Cyanine CyTM5-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories, 715-175-150; IF 1:250), and Cyanine CyTM5-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, 711-175-152; IF 1:250).

Techniques: Affinity Purification, Purification, Western Blot, Control, Fluorescence, Expressing, Microscopy

Journal: Scientific Reports

Article Title: Cardamonin inhibits the growth and stemness of osteosarcoma stem cells by inducing autophagy and inhibiting the Wnt/β-catenin signaling pathway

doi: 10.1038/s41598-025-14209-3

Figure Lengend Snippet: CDN induces autophagy in OS cells. (A) WB detected the expression levels of autophagy marker proteins LC3, SQSTM1, Beclin1, and Atg5 after treating the cells for 24 h as directed. (B) As shown in (A), Immunofluorescence manifests the existence of endogenous LC3 puncta after cell treatment. Scale bar: 25 μm. (C) The WB technique was used to ascertain the LC3 and SQSTM1 expression levels after treating the cells with or without CQ. The significance levels are * p < 0.05 and *** p < 0.001.

Article Snippet: All reagents and antibodies used in this study were purchased from suppliers in China (in brackets): CDN (MCE, HY-N0279), Chloroquine (Sigma, C6628), dimethyl sulfoxide (DMSO, Sigma, 67-68-5), Cell Counting Kit-8 (CCK-8, MCE, HY-K0301), AD-mRFP-GFP-LC3 (Hanbio, HB-AP210 000), hESC-Qualified Matrix, LDEV-free (Corning, 354277), BML284 (MCE, HY-19987), CD133 (Abcam, ab19898), SOX2 (Abcam, ab97959), Nanog (Abcam, ab21624 ), Oct4 (Abcam, ab19857), Beclin1 (Abcam, ab62557), Atg5 (Abcam, ab108327), Wnt1 (Abcam, ab15251), β-catenin (Abcam, ab32572), and SQSTM1 (Origene, UM570012), LC3 (Proteintech, 14600-1-AP) and Ki67(Proteintech, 27309-1-AP).

Techniques: Expressing, Marker, Immunofluorescence

Journal: Veterinary research

Article Title: Foot-and-mouth disease virus activates glycolysis and hijacks HK2 to inhibit innate immunity and promote viral replication.

doi: 10.1186/s13567-025-01497-w

Figure Lengend Snippet: Fig. 4 HK2 enhances FMDV replication through autophagy. A PK-15 cells were infected with FMDV for 12 h, and the number of endogenous LC3 puncta was determined by confocal microscopy. B PK-15 cells were transfected with dual fluorescence-tagged mRFP-eGFP-LC3 for 24 h, followed by FMDV infection for 12 h. Changes in the fluorescence of mRFP-eGFP-LC3 were observed by confocal microscopy. C PK-15 cells were transfected with pEGFP-C1 or pEGFP-HK2 for 24 h, followed by FMDV infection for 12 h. The number of endogenous LC3 puncta was determined by confocal microscopy. D PK-15 cells were co-transfected with dual fluorescence-tagged mRFP-eGFP-LC3 and pCAGGS-HA or with dual fluorescence-tagged mRFP-eGFP-LC3 and pCAGGS-HA-HK2 for 24 h, followed by FMDV infection for 12 h. Changes in the fluorescence of mRFP-eGFP-LC3 were observed by confocal microscopy. E and F PK-15 cells overexpressing pCAGGS-HA or pCAGGS-HA-HK2 were treated or not treated with bafilomycin A1 or CQ for 4 h and then infected with FMDV for 12 h. Western blotting was used to evaluate the protein expression of HA, VP1, P62, LC3B and β-actin (E), and FMDV 3Dpol mRNA levels were measured by qRT‒PCR (F).

Article Snippet: The primary antibodies used in this study were as follows: rabbit polyclonal anti-HK2 (Proteintech, 2A11C3), rabbit polyclonal anti-FMDV VP1 (Bioss, bs-41049R), mouse monoclonal anti-p62 (Cell Signaling Technology, 8025), rabbit polyclonal anti-LC3B (Cell Signaling Technology, 2775), and mouse monoclonal anti-β-actin (Abmart, TP70573) antibodies.

Techniques: Infection, Confocal Microscopy, Transfection, Fluorescence, Western Blot, Expressing

Journal: Veterinary research

Article Title: Foot-and-mouth disease virus activates glycolysis and hijacks HK2 to inhibit innate immunity and promote viral replication.

doi: 10.1186/s13567-025-01497-w

Figure Lengend Snippet: Fig. 7 HK2 utilizes autophagy to degrade IRF3/IRF7 during FMDV infection. A PK-15 cells were co-transfected with pEGFP-HK2 and pCAGGS-HA-IRF3/IRF7 and pRFP-LC3 or with pEGFP-C1 and pCAGGS-HA-IRF3/IRF7 and pRFP-LC3 for 24 h, infected with FMDV for 12 h, fixed, stained with antibodies against HA (blue), GFP (green) and RFP (red), and then examined by confocal microscopy. B and C PK-15 cells were pretreated with the autophagy activator rapamycin (100 nM) or the inhibitors CQ (20 μM) and bafilomycin A1 (0.4 μM) for 4 h and then co-transfected with pEGFP-HK2 and pCAGGS-HA-IRF3/IRF7 for 24 h, followed by FMDV infection for 12 h. Western blotting was used to evaluate the protein expression of GFP, HA, VP1, p62, LC3B and β-actin. D PK-15 cells were transfected with pEGFP-HK2 after 4 h of pretreatment with the autophagy activator rapamycin (100 nM) or the inhibitors CQ (20 μM) and bafilomycin A1 (0.4 μM). The protein expression of GFP, IRF3, IRF7, VP1, p62, LC3B and β-actin was detected by western blotting. E PK-15 cells were co-transfected with pEGFP-HK2 and Flag-p62, EGFP-C1 and Flag-p62, EGFP-HK2 and p3 × Flag-CMV, or EGFP-C1 and p3 × Flag-CMV for 24 h, fixed, stained with DAPI (blue) and antibodies against GFP (green) and Flag (red), and then examined by confocal microscopy.

Article Snippet: The primary antibodies used in this study were as follows: rabbit polyclonal anti-HK2 (Proteintech, 2A11C3), rabbit polyclonal anti-FMDV VP1 (Bioss, bs-41049R), mouse monoclonal anti-p62 (Cell Signaling Technology, 8025), rabbit polyclonal anti-LC3B (Cell Signaling Technology, 2775), and mouse monoclonal anti-β-actin (Abmart, TP70573) antibodies.

Techniques: Infection, Transfection, Staining, Confocal Microscopy, Western Blot, Expressing