Journal:

Article Title: Activation of Protein Kinase C? Induces Serine Phosphorylation of VAMP2 in the GLUT4 Compartment and Increases Glucose Transport in Skeletal Muscle

doi: 10.1128/MCB.21.22.7852-7861.2001

Figure Lengend Snippet: Effects of overexpression of PKCζ on distribution of GLUTs. (A) GLUT4 distribution. PKC isoforms were overexpressed as for Fig. ​Fig.2A.2A. Sixteen hours postinfection, control (CON) cells and cells overexpressing PKCζ or PKCα were either untreated or treated with insulin (INS) for 30 min and were fractionated to plasma membrane (P.M.), internal membrane (I.M.), and very-light-density microsome (V.L.D.M.) fractions. The samples were subjected to SDS-PAGE and were immunoblotted (IB) with anti-GLUT4 antibody. The blot shown is representative of five separate experiments. The graph represents the densitometry measurements (mean ± standard error) of five Western blot analyses. Empty bars, plasma membrane; black-bars, internal membrane (I.M.); gray bars, very-light-density micosomes. (B) Western blots of the distribution of GLUT1 (upper set) and GLUT3 (lower set). PKC isoforms were overexpressed and the cells were treated as for panel A. Following treatment, cells were fractionated to plasma membrane (P.M.) or internal membrane (I.M.) fractions.

Article Snippet: Antibodies against various proteins were obtained from the following sources: anti-GLUT1 and anti-GLUT4 (polyclonal antibodies) were a gift from S. Cushman (Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health); anti-PKC antibodies were purchased from Santa Cruz Biotechnology (polyclonal; Santa Cruz, Calif.) and from Transduction Laboratories (monoclonal; Lexington, Ky.); anti-PI 4-kinase, anti-VAMP2, and anti-syntaxin 4 were purchased from UBI; antiphosphoserine was purchased from Zymed (South San Francisco, Calif.); and horseradish peroxidase and anti-rabbit and anti-mouse immunoglobulin G were obtained from Bio-Rad.

Techniques: Over Expression, SDS Page, Western Blot

Journal: Cell reports methods

Article Title: In situ microwave fixation provides an instantaneous snapshot of the brain metabolome.

doi: 10.1016/j.crmeth.2023.100455

Figure Lengend Snippet: Figure 5. Immunofluorescent analysis of GLUT2 and PC expression in CTX, HIPP, and BS in a mouse model of T1DM (A–C) 20 mm coronal sections of the mouse brain were stained with GLUT2 (green) and NeuN (red) and quantified in in the CTX (A), HIPP (B), and brainstem (BS) (C). Zoomed-in images are shown for the retro-splenial (RS) cortex, Cornu Ammonis 3 (CA3) of HIPP, and the DVC of BS. (D) Quantification of % positive pixels for GLUT2 using HALO software. (E–G) 20 mm coronal sections of the mouse brain were stained with pyruvate carboxylase (PC) (green) and NeuN (red) and quantified in in the CTX (E), HIPP (F), and BS (G). Zoomed-in images are shown for the RS, the CA3 of HIPP, and the DVC of BS. (H) Quantification of percentage of positive pixels for PC using HALO software. Values are presented as mean ± SEM (n = 4 biological replicates), *p < 0.05, **p < 0.01, ***p < 0.001, analyzed by Student’s t test. Scale bars are either 1 mm, 500 mm, or 100 mm. PC, pyruvate carboxylase; GLUT2, glucose transporter 2.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies GLUT1, Rabbit Abcam Cat# ab115730; RRID: AB_10903230 GLUT2, Rabbit Novus Cat# NBP2-22218; RRID: AB_2335858 GLUT3, Rabbit Alomone Cat# AGT-023; RRID:AB_2756644 Pyruvate Carboxylase, Rabbit GeneTex GTX121987 Pyruvate Dehydrogenase (ser 293), Rabbit EMD Milipore Cat# AP1062; RRID:AB_2863934 NeuN, Mouse Abcam ab279295 NeuN, Rabbit Abcam Cat# ab236870; RRID:AB_2927651 GFAP, Chicken GeneTex Cat# GTX85454; RRID:AB_10621124 DAPI Novus NBP2-31156 Goat anti-chicken, Alexa 594 Thermo Fisher Scientific Cat# A-11042; RRID:AB_2534099 Goat anti-rabbit, Alexa 488 Thermo Fisher Scientific Cat# A-11034; RRID:AB_2576217 Goat anti-mouse, Alexa 647 Thermo Fisher Scientific Cat# A-21236; RRID:AB_2535805 Chemicals, peptides, and recombinant proteins U-13C6-glucose, 99% Cambridge Isotope Laboratories CLM-1396-MPT-PK L-Norvaline Sigma-Aldrich N7627-5G Methoxyamine hydrochloride Sigma-Aldrich 226904-25G Pyridine Thermo Fisher Scientific TS-27530 MSTFA +1 Percent TMCS Sigma-Aldrich 375934-10X1ML Hydrochloric Acid Thermo Fisher Scientific HPLC Methanol Thermo Fisher Scientific 34860-4X4L-R PBS Sigma-Aldrich P3813 Heparin Sodium (10,000units/mL) N/A NDC# 25021040010 4% Paraformaldehyde in PBS Thermo Fisher Scientific J19943.K2 Sucrose Thermo Fisher Scientific 036508.A1 Normal Goat Serum Thermo Fisher Scientific 31873 Triton X- Sigma-Aldrich X100 Tween 20 Sigma-Aldrich P1379 Prolong Glass Thermo Fisher Scientific P36984 Streptozotocin Sigma-Aldrich 572201 Citric Acid Thermo Fisher Scientific A940 Sodium citrate dihydrate Thermo Fisher Scientific BP327 2-methylbutane Thermo Fisher Scientific 019387AP Ethanol Decon Labs CAS#: 64-17-5 OCT compound Thermo Fisher Scientific 23-730-571 Deposited data Raw and processed metabolomics data This study Metabolomics Workbench ST002524 and ST002525 Software and algorithms HALO Software Indica Labs N/A Prism Graphpad N/A IsoCorrectoR Heinrich et al.80 N/A DEXSI software Dagley and McConville81 N/A Metaboanalyst Xia et al.82 (https://chemdata.nist.gov/dokuwiki/doku. php?id=chemdata:amdis) (Continued on next page) e1 Cell Reports Methods 3, 100455, April 24, 2023

Techniques: Expressing, Staining, Software

Journal: Cell reports. Medicine

Article Title: Metabolic classification suggests the GLUT1/ALDOB/G6PD axis as a therapeutic target in chemotherapy-resistant pancreatic cancer.

doi: 10.1016/j.xcrm.2023.101162

Figure Lengend Snippet: Figure 4. The GLUT1/ALDOB/G6PD axis drives glucose metabolic reprogramming in glucomet-PDAC (A) Relative G6PD enzyme activity in representative organoids of glucomet-PDAC (n = 6) and lipomet-PDAC (n = 6). (B) Relative G6PD enzyme activity in vector- and ALDOB-overexpressing organoids (n = 3). (C) Relative abundance of oxidative PPP metabolites in control and ALDOB-overexpressing organoids (n = 3).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies GLUT1 abcam Cat#ab115730; RRID:AB_10903230 ALDOA proteintech Cat#11217-1-AP; RRID:AB_2224626 ALDOB proteintech Cat#18065-1-AP; RRID:AB_2273968 ALDOC proteintech Cat#14884-1-AP; RRID:AB_2226691 G6PD abcam Cat#ab993; RRID:AB_296714 a-tubulin proteintech Cat#11224-1-AP; RRID:AB_2210206 actin abmart Cat#M20011; RRID:AB_2936240 Biological samples Human tissue samples for sequencing analyses This study N/A Human blood samples for sequence analyses This study N/A Chemicals, peptides, and recombinant proteins Advanced DMEM/F12 Gibco Cat#12634010 HEPES Gibco Cat#15630080 GlutMAX Gibco Cat#35050061 Penicillin-Streptomycin Gibco Cat#15140122 Primocin InvivoGen Cat#ant-pm-2 B27 supplement Gibco Cat#17504044 Wnt3A conditioned medium Gao lab N/A R-spondin conditioned medium Gao lab N/A Noggin conditioned medium Gao lab N/A Nicotinamide SIGMA Cat#N0636-100G EGF Invitrogen Cat#PHG0313 FGF10 PEPRO TECH Cat#K2717 N-acetyl-L-cysteine SIGMA Cat#A9165-100G A83-01 TOCRIS Cat#2939 Y-27632 Selleck chemicals Cat#S1049 Forskolin Selleck chemicals Cat#S2449 TrypLETM Express Gibco Cat#12605028 Collagenase Type II Gibco Cat#17101015 Matrigel Matrix (For organoid culture) CORNING Cat#356231 Matrigel Matrix (For xenograft) CORNING Cat#354234 Gemcitabine TargetMol Cat#T0251 Paclitaxel Selleck chemicals Cat#S1150 5-fluorouracil SIGMA Cat#F6627 Oxaliplatin Selleck chemicals Cat#S1124 Irinotecan Selleck chemicals Cat#S1198 BAY-876 Selleck chemicals Cat#S8452 6AN Selleck chemicals Cat#S9783 MLN8054 TargetMol Cat#T6315 Alisertib Selleck chemicals Cat#S1133 Uridine Sigma Cat#U3003 Cytidine Sigma Cat#C4654 (Continued on next page) e1 Cell Reports Medicine 4, 101162, September 19, 2023

Techniques: Activity Assay, Plasmid Preparation, Control