Journal: PLoS ONE

Article Title: Effect of Fatty Acids on Human Bone Marrow Mesenchymal Stem Cell Energy Metabolism and Survival

doi: 10.1371/journal.pone.0120257

Figure Lengend Snippet: A) Representative western blots, B) Lactate dehydrogenase A (LDHA), C) Phosphoglycerate mutase 1 (PGAM1), D) Hypoxia inducible factor 1α (HIF1α), E) Isocitrate dehydrogenase (IDH), F) phospho Acetyl CoA carboxylase (ACC)/ACC protein expression. n = 5–6 Protein expression was measured in human bone marrow mesenchymal stem cells (BMMSCs) treated for 24 hr with indicated treatments. The BSA group was treated with media supplemented with 0.55 mM albumin. All fatty acid treated groups were also treated with media supplemented with 0.55 mM albumin in addition to the type and amount of fatty acid indicated in the figures. Values are shown as mean ± SEM.

Article Snippet: Membranes were then blocked for 1 hr in 5% non-fat dry milk (NFDM) in TBST, probed overnight at 4°C with primary antibody, rinsed 4 x 5 min in TBST, probed with appropriate secondary antibody for 1 hr at room temperature, and then rinsed in TBST 4 x 5 min. Primary antibodies included HIF1α (Novus Biologics, NB100–105), LDH-A (Santa Cruz sc-27230), IDH (Abcam, ab36329), phosphoglycerate mutase 1 (PGAM1) (Cell Signaling 7534S), cyclin D1 (Cell Signaling 2922), phospho Rb S780 (Cell Signaling 9307S), phospho acetyl CoA carboxylase (ACC) (Millipore 07–303), and ACC (Jackson 016–050–084).

Techniques: Western Blot, Expressing

Journal: PLOS ONE

Article Title: Helicase-like transcription factor (HLTF) -deleted CDX/TME model of colorectal cancer increased transcription of oxidative phosphorylation genes and diverted glycolysis to boost S-glutathionylation in lymphatic intravascular metastatic niches

doi: 10.1371/journal.pone.0291023

Figure Lengend Snippet: Source, application and concentration of antibodies.

Article Snippet: Rabbit polyclonal HLTF antibody (NBP1-83256) Novus Biologicals (1:100) Rabbit polyclonal CYTB (55090-1-AP) ThermoFisher Scientific (1:50) Rabbit polyclonal PGAM1 (16126-1-AP) ThermoFisher Scientific (1:50) Rabbit polyclonal ANXA1 (71–3400) ThermoFisher Scientific (1:20) Rabbit monoclonal γH2AX-phospho S139 (ab81299) Abcam (1:50) Rabbit monoclonal ENO1 (ab227978) Abcam (1:2,000) Rabbit recombinant monoclonal mouse-specific PDPN (MA5-29742) ThermoFisher Scientific (1:500) , IHC-P: Vector Laboratories RTU Biotinylated Goat Anti-rabbit IgG (H+L) (BP-9100-50).

Techniques: Concentration Assay, Recombinant, Plasmid Preparation

Journal: PLOS ONE

Article Title: Helicase-like transcription factor (HLTF) -deleted CDX/TME model of colorectal cancer increased transcription of oxidative phosphorylation genes and diverted glycolysis to boost S-glutathionylation in lymphatic intravascular metastatic niches

doi: 10.1371/journal.pone.0291023

Figure Lengend Snippet: Summary of site-specific S-glutathionylation identified by 2D-DIGE MALDI-TOF/TOF mass spectrometry. Pr-SSG in HLTF -/- CDX/TME.

Article Snippet: Rabbit polyclonal HLTF antibody (NBP1-83256) Novus Biologicals (1:100) Rabbit polyclonal CYTB (55090-1-AP) ThermoFisher Scientific (1:50) Rabbit polyclonal PGAM1 (16126-1-AP) ThermoFisher Scientific (1:50) Rabbit polyclonal ANXA1 (71–3400) ThermoFisher Scientific (1:20) Rabbit monoclonal γH2AX-phospho S139 (ab81299) Abcam (1:50) Rabbit monoclonal ENO1 (ab227978) Abcam (1:2,000) Rabbit recombinant monoclonal mouse-specific PDPN (MA5-29742) ThermoFisher Scientific (1:500) , IHC-P: Vector Laboratories RTU Biotinylated Goat Anti-rabbit IgG (H+L) (BP-9100-50).

Techniques: Mass Spectrometry

Journal: PLOS ONE

Article Title: Helicase-like transcription factor (HLTF) -deleted CDX/TME model of colorectal cancer increased transcription of oxidative phosphorylation genes and diverted glycolysis to boost S-glutathionylation in lymphatic intravascular metastatic niches

doi: 10.1371/journal.pone.0291023

Figure Lengend Snippet: Light micrographs show positive immunostaining for PDPN (A), a lymphatic-specific endothelial cell marker, in companion with PGAM1 (B), ENOA1 (C) and ANXA1 (D) in tumor cells. 40X magnification. ANXA1 (E) and PGAM1 (F) colocalize (G) in tumor and endothelial cells in lymphatic intravascular niches. A polarized extravascular tumor cell with redistribution of ANXA1 and PGAM1 into a posterior uropod protrusion (yellow insets) is engaged in amoeboid migration, a hallmark of rapid migration . The maximal projection of confocal images obtained from a z stack (500 nm slice) using a 60x objective (oil) are shown. Overlay images were obtained with transmitted light and excitation at 405 nm (DAPI, blue-violet), 555 nm (ANXA1, green) and 647 nm (PGAM1, red).

Article Snippet: Rabbit polyclonal HLTF antibody (NBP1-83256) Novus Biologicals (1:100) Rabbit polyclonal CYTB (55090-1-AP) ThermoFisher Scientific (1:50) Rabbit polyclonal PGAM1 (16126-1-AP) ThermoFisher Scientific (1:50) Rabbit polyclonal ANXA1 (71–3400) ThermoFisher Scientific (1:20) Rabbit monoclonal γH2AX-phospho S139 (ab81299) Abcam (1:50) Rabbit monoclonal ENO1 (ab227978) Abcam (1:2,000) Rabbit recombinant monoclonal mouse-specific PDPN (MA5-29742) ThermoFisher Scientific (1:500) , IHC-P: Vector Laboratories RTU Biotinylated Goat Anti-rabbit IgG (H+L) (BP-9100-50).

Techniques: Immunostaining, Marker, Migration

Journal: Pharmacological research

Article Title: RHBDF1 deficiency suppresses melanoma glycolysis and enhances efficacy of immunotherapy by facilitating glucose-6-phosphate isomerase degradation via TRIM32.

doi: 10.1016/j.phrs.2023.106995

Figure Lengend Snippet: Fig. 3. RHBDF1 maintains the stability of GPI in melanoma. (A) Western blotting analysis of the glycolytic enzymes′ expression after RHBDF1 knockout or knockdown in B16F10 cells and A375 cells. (B) Immunofluorescence staining of GPI protein in B16F10-EV/R1KO tumors. (Red: GPI, blue: DAPI, scale bar: 50 µm). (C) The correlation of RHBDF1 with GPI, PGAM1 and LDHA at the transcriptional level in GEPIA database. (D) qPCR analysis of GPI, PGAM1 and LDHA levels in B16F10 and A375 cells with or without RHBDF1 expression (n = 3). (E) Immunofluorescence staining of the distribution of GPI-6Myc in lysosomes in B16F10-R1KO cells. GPI-6Myc was transfected into the B16F10-R1KO cells for 24 h and the cells were treated with NH4CI for 6 h before staining, the white arrows indicate the co- location of GPI and the lysosome marker, LAMP1 (Red: Myc, green: LAMP1, blue: DAPI, scale bar: 25 µm). (F, G) Western blotting analysis of GPI protein levels in B16F10 and A375 cells when treated with NH4Cl 20 mmol/L or 3-Methyladenine (3-MA) 5 mmol/L for the indicated time. Error bars were represented as mean ± SD, two tailed Student′s t test was used for significance tests between two groups, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Primary antibodies for Myc tag (16286–1-AP), HA tag (51064–2-AP and Chromotek, 7C9–20), PGAM1 (16126–1-AP), CD107a/LAMP1 (APC-65050) and heavy chain of rabbit IgG (P66467–1-lg) were purchased from Proteintech (Wuhan, China).

Techniques: Western Blot, Expressing, Knock-Out, Knockdown, Immunofluorescence, Staining, Transfection, Marker, Two Tailed Test

Journal: Nature chemical biology

Article Title: Bisphosphoglycerate mutase controls serine pathway flux via 3-phosphoglycerate

doi: 10.1038/nchembio.2453

Figure Lengend Snippet: a) Schematic depicting phosphoryl-transfer step between 3-PG, 2-PG and PGAM1. b) Western blot analysis of different mammalian cell lysates using an α-pHis antibody. Top panel shows untreated lysates and bottom panel shows lysates treated with hydroxylamine prior to Western blot analysis (see for Coomassie stain loading control). c) LC-MS analysis of 2,3-BPG levels in wt and BPGM knockout HEK 293T cells (n = 3, mean ± s.d., * = p<0.05). d) Western blot analysis of wt and BPGM knockout HEK 293T cells using an α–pHis or α–PGAM1 antibody (α–actin antibody was used as a loading control). e) Western blot analysis of wt and BPGM knockout HCT116 or MDA-MB-231 cells using an α–pHis or α–PGAM1 antibody (α–actin antibody was used as a loading control). f) LC-MS analysis of 2,3-BPG levels in wt and BPGM knockout HCT116 cells (n = 3, mean ± s.d., *** = p<0.001). See for full Western blot images.

Article Snippet: Anti-β-actin mouse monoclonal antibody (ab8226) and anti-PGAM1 rabbit monoclonal antibody (ab129191) were from Abcam.

Techniques: Western Blot, Staining, Liquid Chromatography with Mass Spectroscopy, Knock-Out

Journal: Nature chemical biology

Article Title: Bisphosphoglycerate mutase controls serine pathway flux via 3-phosphoglycerate

doi: 10.1038/nchembio.2453

Figure Lengend Snippet: a) Western blot analysis of PGAM1 overexpression lysates from wt and BPGM knockout HEK 293T cell lysates using an α–pHis and α–PGAM1 antibody (α–actin antibody was used as a loading control). b) Western blot analysis of a PGAM1 phosphorylation time course in the presence of 2,3-BPG or 1,3-BPG (membranes were stained with Coomassie to serve as a loading control). c) 1,3-BPG-treated PGAM1 was subjected to trypsinization and LC-MS analysis. Shown is the MS/MS spectrum of the tryptic peptide of PGAM1 containing the pHis site (His-11) and in mirror image is shown the MS/MS spectrum of 2,3-BPG induced phosphorylation of PGAM1 to demonstrate that both 1,3-BPG and 2,3-BPG result in His-11 phosphorylation. d) LC-MS analysis of PGAM1 phosphorylation with 1,3-BPG in the absence of 2-PG, low 2-PG (50 μM), or high 2-PG (1000 μM). Extracted ion chromatograms for 1,3-BPG and 2,3-BPG (m/z=264.952) are shown for each of the described reaction conditions. See for full Western blot images.

Article Snippet: Anti-β-actin mouse monoclonal antibody (ab8226) and anti-PGAM1 rabbit monoclonal antibody (ab129191) were from Abcam.

Techniques: Western Blot, Over Expression, Knock-Out, Staining, Liquid Chromatography with Mass Spectroscopy, Tandem Mass Spectroscopy

Journal: Nature chemical biology

Article Title: Bisphosphoglycerate mutase controls serine pathway flux via 3-phosphoglycerate

doi: 10.1038/nchembio.2453

Figure Lengend Snippet: a) BPGM activity converts 1,3-BPG to 2,3-BPG for activation of PGAM1 via histidine phosphorylation. b) Upon BPGM disruption, 2,3-BPG levels and PGAM1 protein and phosphorylation levels decrease without impacting overall glycolysis likely because of 1,3-BPG mediated phosphorylation of PGAM1. Furthermore, as a result of decreased PGAM1 protein and phosphorylation levels, 3-PG, phosphoserine, and serine metabolite levels increase.

Article Snippet: Anti-β-actin mouse monoclonal antibody (ab8226) and anti-PGAM1 rabbit monoclonal antibody (ab129191) were from Abcam.

Techniques: Activity Assay, Activation Assay