rat anti mouse monoclonal f4 80  (Bio-Rad)

Journal: Science Advances

Article Title: Harnessing skin-resident γδ T cells for immunotherapy in cutaneous squamous cell carcinoma

doi: 10.1126/sciadv.aec7215

Figure Lengend Snippet: ( A ) Expression of indicated chemokine receptors by in vitro expanded, live gated CD3 + Vδ1 + γδ T cells. Blood-derived αβ T cells were used as staining control. ( B ) As (A), graphical summary of the percentage of Vδ1 + γδ T cell expressing the chemokine receptors ( n = 5 skin donors). Error bars represent mean ± SD. ( C ) 10 × 10 6 human skin–derived T cells, containing approximately 7% of Vδ1 + γδ T cells, were injected intravenously (i.v.) into NSG mice carrying a xSCC of a volume ranging from 100 to 200 mm 3 [reached approximately 60 to 80 days (d) post–i.d. injection of SCC-13 cells]. Each mouse was injected intraperitoneally (i.p.) with recombinant IL-2 and IL-15 daily until the harvest day. Figure created in BioRender. I. Gratz (2026) https://BioRender.com/zmnd28b . ( D ) Representative plot of the percentage of Vδ1 + γδ T cells engrafted in the spleen, blood, xSCC and murine skin 2, 7, or 14 days posttransfer. ( E to G ) Bar graphs show the absolute numbers of ingoing Vδ1 + γδ T cells, Vδ1 + γδ T cells engrafting spleen and blood normalized to mouse weight (grams), and xSCC and murine skin normalized to tissue weight (grams). (E) n = 7 mice per group; pool of two independent experiments. (F) n = 12 mice per group; pool of four independent experiments; (G) n = 12 mice per group; pool of two independent experiments. Each symbol represents one skin donor. Error bars represent mean ± SD. Statistical significance was determined using the Kruskal-Wallis test with Dunn’s multiple comparisons test. All data points, including extreme values, are shown. ( H ) Representative immunofluorescent staining of colocalized TCRδ/DAPI in HD skin and xSCC 7 days after γδ transfer. Scale bars, 100 μm. Staining controls are shown in fig. S4.

Article Snippet: Human skin–derived T lymphocytes were labeled with Cell Proliferation Dye eFluor 450 (10 μM, Thermo Fisher Scientific, catalog no. 65- 0842-85), washed with PBS, and subsequently stimulated for 6 days with rhuIL-2 (100 IU/ml; BioLegend, catalog no. 589108), rhuIL-15 (20 ng/ml; BioLegend, catalog no. 570603), in addition with mouse anti-human CD3 monoclonal antibody (1 μg/ml; Miltenyi Biotec, catalog no. 130-093-387, RRID:AB_1036144) alone or with rhuIL-1α (9 ng/ml; BioLegend, catalog no. 570004) and rhuIL-18 (9 ng/ml, BioLegend, catalog no. 592102) applied individually or in combination in cTexMacs medium.

Techniques: Expressing, In Vitro, Derivative Assay, Staining, Control, Injection, Recombinant

Journal: Science Advances

Article Title: Harnessing skin-resident γδ T cells for immunotherapy in cutaneous squamous cell carcinoma

doi: 10.1126/sciadv.aec7215

Figure Lengend Snippet: ( A ) Levels of cytokines (pg/mg tissue) produced by HD skin, huSCC, ES, and xSCC. Heatmap bars represent the mean of n = 5 HD skin and huSCC donors, and mean n = 5 of xenograft mice. ( B ) Representative gating strategy and bar graphs of the human skin–derived and ex vivo expanded Vδ1 + γδ T cells expressing IL-1RAcP and IL-18Rα. Peripheral blood αβ T cells and ex vivo expanded skin-derived αβ T cells were used as staining controls. Mean of n = 5 skin donors. ( C ) eFluor450-labeled γδ T cells were cultured under basal conditions [unstimulated or with IL-2 (100 IU/ml) and IL-15 (20 ng/ml)] or stimulated with anti-CD3 (1 μg/ml) and/or IL-1α and IL-18 (9 ng/ml) for 6 days. Proliferation was assessed by the median fluorescence intensity (MFI) of eFluor450 in Vδ1 + by flow cytometry. The representative histograms show the eFluor450 dilution in Vδ1 + T cells in the different conditions. Cell counts were normalized to unit area. ( D ) Bar graphs show the fold change of efluor450 MFI of Vδ1 + treated with anti-CD3, IL-1α, and IL-18 relative to IL-2 and IL-15. Mean of n = 6 skin donors. Statistical analysis was performed using a Friedman test followed by Dunn’s multiple comparisons test. Data in bar graphs (B) and (D) are shown as mean ± SD.

Article Snippet: Human skin–derived T lymphocytes were labeled with Cell Proliferation Dye eFluor 450 (10 μM, Thermo Fisher Scientific, catalog no. 65- 0842-85), washed with PBS, and subsequently stimulated for 6 days with rhuIL-2 (100 IU/ml; BioLegend, catalog no. 589108), rhuIL-15 (20 ng/ml; BioLegend, catalog no. 570603), in addition with mouse anti-human CD3 monoclonal antibody (1 μg/ml; Miltenyi Biotec, catalog no. 130-093-387, RRID:AB_1036144) alone or with rhuIL-1α (9 ng/ml; BioLegend, catalog no. 570004) and rhuIL-18 (9 ng/ml, BioLegend, catalog no. 592102) applied individually or in combination in cTexMacs medium.

Techniques: Produced, Derivative Assay, Ex Vivo, Expressing, Staining, Labeling, Cell Culture, Fluorescence, Flow Cytometry

Journal: Cancer Pathogenesis and Therapy

Article Title: Metabolic pathways and chemotherapy resistance in acute myeloid leukemia (AML): Insights into Enoyl-CoA hydratase domain-containing protein 3 ( ECHDC3 ) as a potential therapeutic target

doi: 10.1016/j.cpt.2025.08.002

Figure Lengend Snippet: Changes in mitochondrial function following ECHDC3 knockdown. (A) TMRE staining results based on ECHDC3 -knockdown cells. siNC cells emitted bright red-orange fluorescence. Cells treated with a mitochondrial membrane-potential disrupter, CCCP, showed very weak or complete absence of red-orange fluorescence. The average fluorescence intensity of the cells was calculated and quantitatively analyzed. (B–C) mtDNA copy number ( MT–CO1 and MT–CO2 ) was quantified via quantitative RT-PCR; (D) Quantitation of mitochondrial SOD activity, wherein SOD activity decreased in ECHDC3 -knockdown cells. (E) Mitophagy biomarkers were detected via western blotting. β-Actin was used as a control. (F–I) Quantitation of the mitophagy pathway protein. Values were presented as mean ± standard error. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. CCCP: Carbonyl cyanide m-chlorophenyl hydrazone; ECHDC3 : Enoyl-CoA hydratase domain-containing protein 3; mtDNA: Mitochondrial DNA; RT-PCR: Real-time polymerase chain reaction; SOD: Superoxide dismutase; TMRE: Tetramethyl rhodamine ethyl ester.

Article Snippet: Western blotting was performed to determine the expression of mitochondrial proteins, using the Mitophagy Antibody Sampler Kit (Cat# 43110, Cell Signaling Technology [CST], MA, USA) and an anti-β-actin mouse monoclonal antibody (Cat# 3700, CST, MA, USA).

Techniques: Knockdown, Staining, Fluorescence, Membrane, Quantitative RT-PCR, Quantitation Assay, Activity Assay, Western Blot, Control, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

Journal: Journal of Translational Medicine

Article Title: PD-1 H (VISTA) drives immunosuppressive reprogramming of glioma-associated myeloid cells to promote glioma progression

doi: 10.1186/s12967-026-08497-0

Figure Lengend Snippet: PD-1 H regulates GAM polarization to promote glioma malignancy and suppress T-cell immunity. ( A ) Flow cytometric analysis of CD206 expression in BV2 cells, used as an in vitro model of GAMs, with PD-1 H knockout (PD-1 H-KO and PD-1 H-KO#1) or negative control (PD-1 H-NC) under basal conditions (blank) or following IL-4 stimulation (M2) for 48 h. ( B ) Flow cytometric analysis of CD86 expression in PD-1 H-deficient or control BV2 cells under basal conditions (blank) or after IFN-γ stimulation (M1) for 48 h. ( C ) Phagocytic activity of PD-1 H-deficient and control BV2 cells, assessed by uptake of fluorescent microspheres (FluoSpheres) after 4 h incubation. ( D ) Cell viability of GL261 glioma cells cultured with conditioned media derived from PD-1 H-NC, PD-1 H-KO, PD-1 H-KO#1, lenti-NC, or PD-1 H-overexpressing BV2 cells, measured by CCK-8 assay at 24, 48, and 72 h (*** p < 0.001, **** p < 0.0001). ( E, F ) Transwell invasion assays of GL261 cells co-cultured with BV2 cells expressing different levels of PD-1 H. Representative images ( E ) and quantification of invading cells per field ( F ) Are shown (* p < 0.05). ( G, H ) Colony formation assay of GL261 cells cultured with conditioned medium from BV2 cells as indicated. Representative images ( G ) and quantification of colony numbers ( H ) are shown (* p < 0.05, ** p < 0.01). ( I, J ) Wound-healing assays of GL261 cells at 0 h and 48 h after scratch following treatment with BV2 cell–derived conditioned media. Representative images ( I ) and quantification of wound closure ( J ) Are shown (* p < 0.05, ** p < 0.01). ( K ) Antigen-specific proliferation of OT-I CD8+ T cells in the priming stage. Total lymph node cells were stimulated with OVA257-268 peptide and co-cultured with BV2 cells for 48 h. Representative CFSE histograms are shown. ( L ) Proliferation analysis of CD8+ T cells in the differentiation stage. Pre-primed T cells were co-cultured with BV2 cells under IL-2 stimulation for another 48 h. Relative proliferative capacity was quantified via derivative analysis of normalized CFSE signals. Data are presented as mean ± SD (** p < 0.01)

Article Snippet: Mice were subsequently treated with PBS or anti-PD-1 H monoclonal antibody (13F3, 300 μg/mouse, HY-P990145, MCE) every two days, a dosing regimen based on previous studies [ , ].

Techniques: Expressing, In Vitro, Knock-Out, Negative Control, Control, Activity Assay, Incubation, Cell Culture, Derivative Assay, CCK-8 Assay, Colony Assay

Journal: Journal of Translational Medicine

Article Title: PD-1 H (VISTA) drives immunosuppressive reprogramming of glioma-associated myeloid cells to promote glioma progression

doi: 10.1186/s12967-026-08497-0

Figure Lengend Snippet: PD-1 H expression and distribution in glioma. ( A ) Representative IHC images of PD-1 H in human glioma vs. normal brain tissue (scale bars, 200 μm). ( B ) Quantification of IHC scores for PD-1 H in glioma ( n = 27) and normal brain ( n = 5) samples (** p < 0.01). ( C ) Violin plots showing expression distribution of PD-1 H and other immune checkpoint molecules (TIM-3, LAG-3, TIGIT, CTLA-4, PD-L1, and PD-1) in glioma samples from the TCGA dataset. ( D ) Bulk RNA-seq of PD-1 H expression levels in glioma ( n = 702) and normal brain ( n = 423) samples based on RNA-seq data from TCGA and GTEx datasets (**** p < 0.0001). ( E ) UMAP plot showing single-cell RNA-seq data of glioma, colored by cell type (5.27 × 10 5 cells from 85 glioma samples, single-cell portal, SCP2389). ( F ) UMAP plot with PD-1 H expression intensity (color scale). ( G ) Bar chart showing the expression of PD-1 H in different cell clusters

Article Snippet: Mice were subsequently treated with PBS or anti-PD-1 H monoclonal antibody (13F3, 300 μg/mouse, HY-P990145, MCE) every two days, a dosing regimen based on previous studies [ , ].

Techniques: Expressing, RNA Sequencing, Single Cell

Journal: Journal of Translational Medicine

Article Title: PD-1 H (VISTA) drives immunosuppressive reprogramming of glioma-associated myeloid cells to promote glioma progression

doi: 10.1186/s12967-026-08497-0

Figure Lengend Snippet: PD-1 H expression in GAMs is associated with immune cell composition and clinical outcome in glioma. ( A–E ) Representative IHC staining of PD-1 H, CD68, CD163, CD80, and CD8 in a glioma specimen with low PD-1 H expression (patient-1). ( F–J ) Corresponding IHC staining of PD-1 H, CD68, CD163, CD80, and CD8 in a glioma specimen with high PD-1 H expression (patient-2). Scale bars, 200 μm. ( K–N ) Correlation analyses between PD-1 H and immune cell markers, including CD68, CD163, CD80 and CD8 in glioma samples ( n = 27). ( O–R ) Correlation analyses of expression levels between PD-1 H and immune checkpoint molecules, including TIM-3, PD-L1, PD-1, and CTLA-4 in glioma samples from the TCGA dataset ( n = 702, Spearman correlation, p < 0.001). ( S, T ) Kaplan–Meier survival curves of glioma patients stratified by PD-1 H expression levels in GAMs, including progression-free survival (PFS, S) and overall survival (OS, T) (Log-rank test)

Article Snippet: Mice were subsequently treated with PBS or anti-PD-1 H monoclonal antibody (13F3, 300 μg/mouse, HY-P990145, MCE) every two days, a dosing regimen based on previous studies [ , ].

Techniques: Expressing, Immunohistochemistry

Journal: Journal of Translational Medicine

Article Title: PD-1 H (VISTA) drives immunosuppressive reprogramming of glioma-associated myeloid cells to promote glioma progression

doi: 10.1186/s12967-026-08497-0

Figure Lengend Snippet: PD-1 H expression in GAMs promotes glioma progression and impairs antitumor T-cell responses in vivo. ( A ) Schematic illustration of the in vivo experimental design. C57BL/6 mice were intracranially implanted with GL261-luc glioma cells together with GAMs expressing PD-1 H overexpression (PD-1 H-OE), negative control (PD-1 H-NC), or PD-1 H knockout (PD-1 H-KO). Tumor growth was monitored by bioluminescence imaging at the indicated time points. ( B ) Representative bioluminescence images of tumor-bearing mice from each group at days 7, 14, and 21 after implantation. ( C ) Quantification of tumor bioluminescence radiance over time in mice receiving PD-1 H-OE, PD-1 H-NC, or PD-1 H-KO GAMs (* p < 0.05, *** p < 0.001). ( D ) Kaplan–Meier survival analysis of glioma-bearing mice in the indicated groups. Statistical significance was determined by log-rank test ( p < 0.001). ( E ) Flow cytometric analysis of GAM phenotypes isolated from glioma tissues. Representative histograms show expression of CD206 and MHC-II on GAMs from PD-1 H-OE, PD-1 H-NC, and PD-1 H-KO groups. ( F, G ) Representative flow cytometry histograms showing expression of PD-1 and TIM-3 on tumor-infiltrating CD8 + T ( F ) and CD4 + T ( G ) cells from each group

Article Snippet: Mice were subsequently treated with PBS or anti-PD-1 H monoclonal antibody (13F3, 300 μg/mouse, HY-P990145, MCE) every two days, a dosing regimen based on previous studies [ , ].

Techniques: Expressing, In Vivo, Over Expression, Negative Control, Knock-Out, Imaging, Isolation, Flow Cytometry

Journal: Journal of Translational Medicine

Article Title: PD-1 H (VISTA) drives immunosuppressive reprogramming of glioma-associated myeloid cells to promote glioma progression

doi: 10.1186/s12967-026-08497-0

Figure Lengend Snippet: PD-1 H regulates transcriptional programs and signaling pathways in GAMs. ( A ) Principal component analysis (PCA) of transcriptomic profiles from BV2 cells with PD-1 H-KO (KO), PD-1 H-NC (NC), or PD-1 H-OE (OE), showing distinct clustering among groups. ( B, C ) Volcano plot ( B ) and heatmap ( C ) showing differentially expressed genes (DEGs) in PD-1 H-OE vs. PD-1 H-NC. Upregulated and downregulated genes are highlighted. ( D, E ) Volcano plot ( D ) and heatmap ( E ) displaying DEGs in PD-1 H-KO vs. PD-1 H-NC. ( F, G ) Gene ontology (GO) enrichment analysis of DEGs from PD-1 H-OE vs. PD-1 H-NC ( F ) and PD-1 H-KO vs. PD-1 H-NC ( G ), with dot size indicating gene count and color representing statistical significance. ( H ) Western blot analysis of NF-κB, phosphorylated AKT (p-AKT), total AKT, in BV2 cells with different expression levels of PD-1 H. The PD-1H-blocking antibody 13F3 was applied as indicated (−/+)

Article Snippet: Mice were subsequently treated with PBS or anti-PD-1 H monoclonal antibody (13F3, 300 μg/mouse, HY-P990145, MCE) every two days, a dosing regimen based on previous studies [ , ].

Techniques: Protein-Protein interactions, Western Blot, Expressing, Blocking Assay

Journal: Journal of Translational Medicine

Article Title: PD-1 H (VISTA) drives immunosuppressive reprogramming of glioma-associated myeloid cells to promote glioma progression

doi: 10.1186/s12967-026-08497-0

Figure Lengend Snippet: Therapeutic blockade of PD-1 H suppresses glioma progression in PD-1 H-/- host mice. ( A ) Schematic illustration of the in vivo experimental design. C57BL/6PD-1 H-/- mice were intracranially implanted with GL261-luc glioma cells together with PD-1 H-NC GAMs. Mice were treated with PBS or anti-PD-1 H monoclonal antibody at the indicated time points, and tumor growth was monitored by serial bioluminescence imaging. ( B ) Representative bioluminescence images of mice treated with PBS or anti-PD-1 H antibody at days 7, 14, 21, and 28 after implantation. ( C ) Quantification of tumor bioluminescence radiance over time in PBS- and anti-PD-1 H-treated mice, * p < 0.05. ( D ) Kaplan–Meier survival curves of glioma-bearing mice receiving PBS or anti-PD-1 H treatment, log-rank test, * p < 0.05

Article Snippet: Mice were subsequently treated with PBS or anti-PD-1 H monoclonal antibody (13F3, 300 μg/mouse, HY-P990145, MCE) every two days, a dosing regimen based on previous studies [ , ].

Techniques: In Vivo, Imaging

Journal: iScience

Article Title: cGAS/STING sensing in dendritic cells discriminates between daptomycin sensitive and resistant Staphylococcus aureus clinical isolates

doi: 10.1016/j.isci.2026.115854

Figure Lengend Snippet: TLR2 is required for MutuDC sensing of, but not internalization of MRSA (A) Relative pHrodo labeled MRSA internalization by MutuDC over 4 h following stimulation with DapS A8819 (light blue symbols) or DapR A8817 (dark blue symbols), or media alone (white squares). Prior to stimulation, MutuDC were pre-treated for 1 h with TLR2 blocking antibody (clone T2.5; triangles with dashed lines) or media alone (circles with filled lines). Relative MRSA internalization by each DC subset is expressed as the gMFI of pHrodo. Results show the mean and (SD) of duplicates from one experiment, representative of two independent experiments. (B) Cytokine secretion (pg/mL) by MutuDC stimulated with TLR2 ligand peptidoglycan of S. aureus (PGN-SA) (10 μg/mL) or (C) DapS (A8819; light blue) or DapR (A8817; dark blue) MRSA (MOI of 10) for 18 h. MutuDC were first pre-treated with either TLR2 blocking antibody (dot-filled bars) or media alone (filled bars) as in A, or an isotype control (clone 163D3, empty bars) at 1 μg/mL. Results pooled from four (B) or three (C) independent experiments and expressed as the mean ± SEM, with each symbol (circle, square, and directional triangles) representing paired experimental replicates ( n = 3). Statistical significance determined using paired t test and reported as indicated by an ∗ when p ≤ 0.05. (D) Expression of surface activation markers by MutuDC stimulated with DapS A8819 MRSA. DC were pre-treated with TLR2 blocking antibody (black trace), isotype control (dashed red trace), and media alone (light blue shaded). Unstained control sample is shown for each marker (black dashed trace). Data shown from one experiment, representative of three independent experiments.

Article Snippet: mAB mTLR2- anti-mouse/human TLR2 , InvivoGen , Cat# mab-mtlr2; RRID: AB_763722.

Techniques: Labeling, Blocking Assay, Control, Expressing, Activation Assay, Marker

Journal: iScience

Article Title: 3-Hydroxypropionic acid converts inflammatory macrophage glycolysis into mitochondrial oxidation through GAPDH carboxyethylation

doi: 10.1016/j.isci.2026.116258

Figure Lengend Snippet: GAPDH is modified with carboxyethylation at Cys 247 (A) Mass spectrometry analysis of GAPDH peptide (234–260) with carboxyethylation and 3-HPA (5 mM) incubated with the GAPDH peptide (234–260) at 37 °C for 4 h. (B) SPR analysis of the affinity of the anti-ceC247 antibody for the carboxyethylation modified GAPDH peptide (234–260) and unmodified GAPDH peptide (234–260). (C) ELISA-based binding curve of anti-ceC247 antibody to modified GAPDH peptide (GAPDH ce (234–260)) and unmodified GAPDH peptide (234–260). Data are the means ± SD and n = 3 per group. Statistical significance was determined using two-way ANOVA followed by ∗∗p < 0.01. (D) Chemical structures of cysteine carboxyethylation and cysteine lactylation. (E) Unmodified GAPDH peptide (234–260), carboxyethylated peptide (GAPDH ce (234–260)), and lactylated peptide (GAPDH lac (234–260)) were tested with the anti-ceC247 antibody in dot blot assays. (F) Immunoblots of lysates from 293 T cells overexpressing GAPDH, which were treated with 5 mM 3-HPA and 5 μM MG132. The blots were probed with the anti-ceC247 antibody. (G) 3-HPA incubated with the GAPDH peptide (234–260) at 37 °C for 4 h. An anti-ceC247 antibody and the anti-wtC247 antibody were used in dot blot assays.

Article Snippet: Subsequently, cells were incubated overnight at 4°C with primary antibodies: rabbit anti-GAPDH polyclonal antibody (R1210-1, HUABIO, 1:200 dilution) and mouse anti-ceC247 monoclonal antibody (1:200 dilution).

Techniques: Modification, Mass Spectrometry, Incubation, Enzyme-linked Immunosorbent Assay, Binding Assay, Dot Blot, Western Blot

Journal: iScience

Article Title: 3-Hydroxypropionic acid converts inflammatory macrophage glycolysis into mitochondrial oxidation through GAPDH carboxyethylation

doi: 10.1016/j.isci.2026.116258

Figure Lengend Snippet: 3-HPA-induced carboxyethylation of GAPDH promotes its degradation through the ubiquitin-proteasome pathway (A) Chemical structure of carboxyethylated cysteine (left) and structures of aspartic acid (D), glutamic acid (E), methionine (M), and cysteine (C). (B) Immunoblot of GAPDH after transient transfection of flag-tagged GAPDH(C), GAPDH(D), GAPDH(M), GAPDH(E) plasmid in 293 T cells at 24 h, 36 h, and 48 h. (C) Immunoblot of GAPDH after CHX treatment. The GAPDH antibody was used to compare the degradation rates of GAPDH(M), GAPDH(E), GAPDH(D), and GAPDH(C). Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test ∗p < 0.05; ns, not significant. (D) Immunoblot and quantitative analysis of GAPDH ce after CHX treatment. The anti-ceC247 antibody and anti-GAPDH antibody were used to compare the degradation rates of carboxyethylated GAPDH. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test ∗∗p < 0.01; ns, not significant. (E) Immunoblot and quantitative analysis of GAPDH ce after 3-HPA treatment. The anti-ceC247 antibody and anti-GAPDH antibody were used to compare the content of carboxyethylated GAPDH and total GAPDH. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test ∗p < 0.05; ∗∗p < 0.01; ns, not significant. (F) Immunoblot and quantitative analysis of GAPDH ce in 293 T cells treated with 3-HPA (5 mM) combined with proteasomal inhibitor MG132, autophagic inhibitor Chloroquine, or lysosomal inhibitor Bafilomycin A1. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test ∗∗p < 0.01; ∗∗∗∗p < 0.0001; ns, not significant. (G) Immunoprecipitation of GAPDH or GAPDH ce followed by immunoblotting for Myc in 293 T cells transfected with Myc-Ub. Cells were treated with 3-HPA (5 mM) and MG132 (5 μM) for 24 h. (H) Immunoprecipitation of GAPDH or GAPDH ce followed by immunoblotting for Myc in 293 T cells transfected with Myc-Ub mutants (K6O, K11O, K27O, K29O, K33O, K48O, K63O). Cells were treated with 3-HPA (5 mM) for 24 h.

Article Snippet: Subsequently, cells were incubated overnight at 4°C with primary antibodies: rabbit anti-GAPDH polyclonal antibody (R1210-1, HUABIO, 1:200 dilution) and mouse anti-ceC247 monoclonal antibody (1:200 dilution).

Techniques: Ubiquitin Proteomics, Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation