ha cat no 3724s  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: ER-to-Golgi Trafficking is a Nutrient-Sensitive Checkpoint Linking Glucose Starvation to Cell Surface Remodeling

doi: 10.1101/2025.10.31.685804

Figure Lengend Snippet: ( a, b ) U2OS cells were transiently transfected with HA-SEC24A, HA-SEC24B, HA-SEC24C, or HA-SEC24D expression constructs as indicated. After 48 h, cells were incubated in Dulbecco’s Modified Eagle Medium (DMEM) with or without glucose for 1 h, followed by anti-hemagglutinin (HA) antibody staining. ( a ) Representative images showing HA-SEC24 redistribution. ( b ) SEC24 puncta number and puncta intensity (mean ± S.E.M., n = 3 biological replicates, >30 cells per replicate). Statistical significance was assessed using a two-tailed paired Student’s t -test. ***P < 0.001, *P < 0.05. Scale bar, 10 μm. A.D.U., analog-to-digital unit. ( c ) U2OS cells were transiently transfected with the E-cadherin RUSH construct and siRNAs targeting SEC24A , SEC24B , SEC24C , or SEC24D , as indicated. After 48 h, representative immunoblot analyses demonstrated the level of knockdown using the respective SEC24 antibodies. ( d ) U2OS cells were transiently transfected with the E-cadherin RUSH construct and either non-targeting control RNAi (siNT) or si SEC24C , with or without RNAi-resistant HA-tagged SEC24C. After 48 h, cells were incubated in DMEM with or without glucose for 1 h, followed by biotin treatment for 30 min. Representative immunofluorescence images following anti-HA and DAPI staining show expression of HA-SEC24C in the cells. EV, empty vector. Scale bar, 10 μm.

Article Snippet: After blocking the membrane with 5% skim milk, blots were probed with antibodies directed against the following targets: ULK1 (Cat. No. 8058, Cell Signaling), p-ULK1 (S555) (Cat. No. 5869, Cell Signaling), ATG7 (Cat. No. 2631, Cell Signaling), ULK2 (Cat. No. HPA009027, Sigma-Aldrich), ATG13 (Cat. No. SAB4200100, Millipore Sigma), p-ATG13 (S318) (Cat. No. 600-401-C49 Rockland), GFP (Cat. No. ab6556, Abcam), FLAG (Cat. No. F1804, Millipore Sigma), HA (Cat. No. 3724, Cell Signaling), GAPDH (Cat. No. G9545, Sigma Aldrich), p-ACC (S79) (Cat. No. 11818, Cell Signaling), ACC (Cat. No. 3676, Cell Signaling), p-S6 (S235/236) (Cat. No. 4858, Cell Signaling), S6 (Cat. No. 2317, Cell Signaling), SEC24C (Cat. No. 8531, Cell Signaling), SEC24A (Cat. No. 15958-1-AP, Proteintech), SEC24B (Cat. No. A304-876A, Bethyl), SEC24D (Cat. No. 14687, Cell Signaling), and RAB9 (Cat. No. 5133, Cell Signaling).

Techniques: Transfection, Expressing, Construct, Incubation, Modification, Staining, Two Tailed Test, Western Blot, Knockdown, Control, Immunofluorescence, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Phosphoglycerate dehydrogenase interacts with and inhibits the protein kinase TAK1 to mitigate septic shock

doi: 10.1016/j.jbc.2025.110658

Figure Lengend Snippet: PHGDH interacts with TAK1. A , immunoprecipitation was performed using RAW264.7 cell extracts with or without LPS stimulation. Exogenous PHGDH-Flag was immunoprecipitated with anti-Flag beads, while endogenous TAK1 was immunoprecipitated with an anti-TAK1 antibody. Immunoblot analysis was then conducted using the indicated antibodies. B , representative immunofluorescence staining images of TAK1 ( green ), and PHGDH ( red ) of PMs, in which nuclei were stained with DAPI ( blue ). Scale bars represent 10 μm. C – F , immunoprecipitation of exogenous PHGDH-HA with anti-HA-agarose ( C ) or TAK1-Flag with anti-Flag beads ( D ) or endogenous PHGDH with an anti-PHGDH antibody ( E ) or endogenous TAK1 with an anti-TAK1 antibody ( F ) and immunoblot analysis with the indicated antibodies were performed using TLR4-293T ( C and D ) or PM ( E and F ) extracts without or with LPS stimulation for 30 min. G , Ctrl-HA or PHGDH-HA and TAK1-Flag were expressed in TLR4-293T cells stimulated with LPS for the indicated times. Immunoprecipitation of exogenous PHGDH-HA with anti-HA-agarose and immunoblot analysis with the indicated antibodies were performed. H , protein levels of p-TAK1 (T184/187), TAK1, PHGDH, and HA in RAW264.7 cells transfected with siCtrl or siPHGDH and/or with the HA-Ctrl, HA-PHGDH, HA-PHGDH aa 1 to 285, or HA-PHGDH aa 285 to 533 plasmid and stimulated with LPS for 30 min were analyzed. I The expression levels of the proinflammatory cytokines Il-1β and Il-6 were measured by qPCR in RAW264.7 cells transfected with siCtrl or siPHGDH and/or with the HA-Ctrl, HA-PHGDH, HA-PHGDH aa 1 to 285, or HA-PHGDH aa 285 to 533 plasmid and subsequently stimulated with LPS for 6 h. J , His pull-down assay results show the direct binding domains of PHGDH and TAK1. Purified GST/His were used as controls. The data are from three independent experiments performed with biological duplicates and are shown as the mean ± SEM ( n = 3) ( I ) or are representative of three independent experiments ( A , C – H , and J ). NS, not significant ( p ≥ 0.05), ∗∗ p < 0.01 and ∗∗∗ p < 0.001; one-way ANOVA. IL, interleukin; LPS, lipopolysaccharide; PHGDH, phosphoglycerate dehydrogenase; PM, peritoneal macrophage; TAK1, transforming growth factor-β–activated kinase 1; TLR4, Toll-like receptor 4.

Article Snippet: After SDS-PAGE, proteins were transferred onto a polyvinylidene fluoride membrane (Bio-Rad), blocked with 5% milk, and probed with specific antibodies diluted in primary antibody dilution buffer (Beyotime), including PHGDH (1:1000, Cat# ab240744, Abcam); TAK1(1:1000, Cat# 5206, CST); TAK1(1:1000, Cat# CY7057, Abways); p-TAK1 (T184/187) (1:1000, Cat# 4508, CST); IKKβ (1:1000, Cat# 8943, CST); p-IKKα/β (S176/180) (1:1000, Cat# 2697, CST); JNK (1:1000, Cat# 9252T, CST); p-JNK (T183/Y185) (1:1000, Cat# 4668T, CST); p38 (1:1000, Cat# 8690T, CST); p-p38 (T180/Y182) (1:1000, Cat# 4511T, CST); ERK(1:2000, Cat# 4695T, CST); p-ERK (T202/Y204) (1:1000, Cat# 4370T, CST); p65 (1:1000, Cat# 8242, CST); HA (1:2000, Cat# 3724S, CST), Flag (1:2000, Cat# F1804, Sigma); His (1:1000, Cat# AH367, Beyotime); GST 1:1000, (1:1000, Cat# 10000-0-AP, Proteintech); Myc (1:1000, Cat# 2278S, CST); cleaved caspase1 (1:1000, Cat# BY9026, Abways); cleaved-caspase 3 p17 (1:1000, Cat# 341034, Zenbio); cleaved-caspase 8 (1:1000, Cat# 250106, Zenbio); actin (1:3000, Cat# sc-47778, Santa Cruz); and tubulin (1:3000, Cat# ab7792, Abcam).

Techniques: Immunoprecipitation, Western Blot, Immunofluorescence, Staining, Transfection, Plasmid Preparation, Expressing, Pull Down Assay, Binding Assay, Purification

Journal: The Journal of Biological Chemistry

Article Title: Phosphoglycerate dehydrogenase interacts with and inhibits the protein kinase TAK1 to mitigate septic shock

doi: 10.1016/j.jbc.2025.110658

Figure Lengend Snippet: PHGDH interacts with TAK1 in a tetrameric form. A , crosslinking with 0.25 mM disuccinimidyl suberate (DSS) and Western blot analysis of PHGDH in PMs pretreated with 15 μM ethyl 5-(2-furoylamino)-3-methyl-4-thiocyanato-2-thiophenecarboxylate (CBR-5884) for 6 h. B , immunoprecipitation of exogenous PHGDH-HA with anti-HA-agarose and immunoblot analysis with the indicated antibodies were performed using TLR4-293T cells pretreated with 15 μM CBR-5884 for 6 h and treated with LPS for 30 min. C , PMs were pretreated with 15 μM CBR-5884 for 6 h and then stimulated with LPS for 6 h. The mRNA levels of the proinflammatory cytokines Il-1β and Il-6 were measured by qPCR. D , TAK1 and Flag expressions were measured by Western blotting ( left panel ) in RAW264.7 cells after transfected with siCtrl or siTAK1 and/or with the Flag-Ctrl-Flag, TAK1-Flag, TAK1-T184/187A-Flag, or TAK1-T184/187D-Flag plasmid. The expression levels of the proinflammatory cytokines Il-1β and Il-6 were measured by qPCR ( right panel ) in RAW264.7 cells transfected with siCtrl or siTAK1 and/or with the Flag-Ctrl-Flag, TAK1-Flag, TAK1-T184/187A-Flag, or TAK1-T184/187D-Flag plasmid, pretreated with 15 μM CBR-5884 for 6 h, and stimulated with LPS for 6 h. E , PHGDH-KO-PMs were transfected with PHGDH-Flag or PHGDH (V425M)-Flag for 48 h, crosslinking was performed with 0.25 mM DSS, and the PHGDH complexes were evaluated by Western blotting. F , immunoprecipitation of exogenous Flag-PHGDH with anti-Flag beads and immunoblot analysis with the indicated antibodies were performed using TLR4-293T cells transfected with the Ctrl-Flag, PHGDH-Flag, or PHGDH(V425M)-Flag plasmids. G and H , RAW264.7 cells were transfected with siCtrl or siPHGDH and/or with the Ctrl-Flag, PHGDH-Flag, or PHGDH(V425M)-Flag plasmids and were then stimulated with LPS. The phosphorylated and total TAK1 protein levels were measured by Western blotting ( G ), and proinflammatory cytokine mRNA expression levels were measured by qPCR ( H ). The data are from three independent experiments performed with biological duplicates and are shown as the mean ± SEM ( n = 3) ( C , D , and H ) or are representative of three independent experiments ( A , B , E , F , and G ). ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; two-tailed Student’s t test. In ( C ), statistical analyses were performed with two-tailed Student’s t test. In ( D ) and ( H ), statistical analyses were performed with one-way ANOVA. IL, interleukin; LPS, lipopolysaccharide; PHGDH, phosphoglycerate dehydrogenase; PM, peritoneal macrophage; TAK1, transforming growth factor-β–activated kinase 1; TLR4, Toll-like receptor 4.

Article Snippet: After SDS-PAGE, proteins were transferred onto a polyvinylidene fluoride membrane (Bio-Rad), blocked with 5% milk, and probed with specific antibodies diluted in primary antibody dilution buffer (Beyotime), including PHGDH (1:1000, Cat# ab240744, Abcam); TAK1(1:1000, Cat# 5206, CST); TAK1(1:1000, Cat# CY7057, Abways); p-TAK1 (T184/187) (1:1000, Cat# 4508, CST); IKKβ (1:1000, Cat# 8943, CST); p-IKKα/β (S176/180) (1:1000, Cat# 2697, CST); JNK (1:1000, Cat# 9252T, CST); p-JNK (T183/Y185) (1:1000, Cat# 4668T, CST); p38 (1:1000, Cat# 8690T, CST); p-p38 (T180/Y182) (1:1000, Cat# 4511T, CST); ERK(1:2000, Cat# 4695T, CST); p-ERK (T202/Y204) (1:1000, Cat# 4370T, CST); p65 (1:1000, Cat# 8242, CST); HA (1:2000, Cat# 3724S, CST), Flag (1:2000, Cat# F1804, Sigma); His (1:1000, Cat# AH367, Beyotime); GST 1:1000, (1:1000, Cat# 10000-0-AP, Proteintech); Myc (1:1000, Cat# 2278S, CST); cleaved caspase1 (1:1000, Cat# BY9026, Abways); cleaved-caspase 3 p17 (1:1000, Cat# 341034, Zenbio); cleaved-caspase 8 (1:1000, Cat# 250106, Zenbio); actin (1:3000, Cat# sc-47778, Santa Cruz); and tubulin (1:3000, Cat# ab7792, Abcam).

Techniques: Western Blot, Immunoprecipitation, Transfection, Plasmid Preparation, Expressing, Two Tailed Test