Table 1.
Immunoblotting conditions.
| Antigen | Blocking | 1° Antibody | 1° Antibody RRID | 2° Antibody | ECL Reagent |
|---|---|---|---|---|---|
| G6PD | 5% BSA | 1:2000 in 5% BSA, cat#8866, lot#3, Cell Signaling Technology, Danvers, MA, USA | AB_10827744 | 1:2000 Rabbit-HRP |
Western Lightning™ |
| GFPT1 | 5% milk | 1:1000 in 5% BSA, cat#14132-1-AP, Proteintech, Rosemont, IL, USA | AB_2110155 | 1:2000 Rabbit-HRP |
Super Signal™ |
| GLUT1 | 5% milk | 1:4000 in 5% BSA, cat#07-1401, lot#2882724, Millipore, St. Louis, MO, USA | AB_1587074 | 1:2000 Rabbit-HRP |
Super Signal™ |
| GLUT3 | 5% milk | 1:5000 in 5% BSA, cat#AB1344, lot#2943583, Millipore, St. Louis, MO, USA | AB_1587078 | 1:2000 Rabbit-HRP |
Western Lightning™ |
| GLUT4 | 5% BSA | 1:2000 in 5% BSA, cat#07-1404, lot#2890837, Millipore, St. Louis, MO, USA | AB_1587080 | 1:2000 Rabbit-HRP |
Western Lightning™ |
| GLUT6 | 5% BSA | 1:1000 in 5% BSA, cat#TA500639, lot#A001, OriGene Technologies, Rockville, MD, USA | AB_2270444 | 1:5000 Mouse-HRP |
Super Signal™ |
| GLUT10 | 5% milk | 1:5000 in 5% BSA, cat#sc-21635, lot#G0214, Santa Cruz Biotechnology, Dallas, TX, USA | AB_10989951 | 1:5000 Goat-HRP |
Western Lightning™ |
| HXII | 5% BSA | 1:1000 in 5% BSA, cat#sc-6521, lot#H2611, Santa Cruz Biotechnology, Dallas, TX, USA | AB_648073 | 1:5000 Goat-HRP |
Western Lightning™ |
| O-GlcNAc | 5% milk | 1:2000 in 5% BSA, cat#9875, lot#4, Cell Signaling Technology, Danvers, MA, USA | AB_10950973 | 1:5000 Mouse-HRP |
Western Lightning™ |
Antibodies and blotting conditions utilized in the immunoblotting analyses. All bovine serum albumin (BSA) and non-fat dry milk solutions were made by dissolving the reagent in a 1× Tris-buffered saline solution containing 0.1% Tween-20. Information on the primary antibodies utilized including dilution, solution diluted in, company name or developer, catalog number, lot number, associated Research Resource Identifiers (RRIDs) or references providing validation data are indicated in the table. The species, and dilution factor for the horseradish peroxidase (HRP) conjugated secondary antibodies are indicated in the table. All HRP-conjugated secondary antibodies were diluted in a 5% BSA, 1× TBS + 0.1% Tween-20 solution. The goat-HRP antibody was cat#PA1-28664, lot# RK2302292 from ThermoScientific (Waltham, MA, USA). The mouse-HRP antibody was cat#12-349, lot# 2365945 from Millipore (Burlington, MA, USA). The rabbit-HRP antibody was cat#PI31460 (lot# RB230194, SE251028, SH253595, and TG266717) from Fisher Scientific (Waltham, MA, USA). The enhanced chemiluminescence (ECL) substrate reagents utilized were either the Western Lightning™ Plus-ECL, cat# NEL105001EA from Perkin Elmer Life Sciences (Waltham, MA, USA) or the Super Signal™ West Femto Chemiluminescent Substrate, cat# PI34096, from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). G6PD = glucose-6-phosphate dehydrogenase; GFPT1 = glutamine-fructose-6-phosphate transaminase 1; GLUT = glucose transporter; O-GlcNAc = O-linked β-N-acetylglucosamine.