Primary antibodies used were mouse-anti-NDPK-B (MC-412, Kamiya, Seattle, WA, USA, 1:1000), rabbit-anti-NDPK-A (sc-343, Santa Cruz, Heidelberg, Germany, 1:500; detects both NDPK-A and NDPK-B in mouse retinae), mouse-anti-Ang-2 (sc-74403, Santa Cruz, Heidelberg, Germany, 1:500), mouse-anti-O-GlcNAc (ab-2739, Abcam, Cambridge, UK, 1:1000), rabbit-anti-OGA (SAB4200267, Sigma, Munich, Germany, 1:1000), rabbit-anti-OGT (O-6264, Sigma, Munich, Germany, 1:1000), rabbit-anti-N1-phosphohistidine (MABS1330, Millipore, Darmstadt, Germany, 1:1000), mouse-anti-γ-tubulin (T6557, Sigma-Aldrich, Munich, Germany, 1:2000), rabbit-anti-GFAT (obtained in cooperation with Weigert, Tübingen), and sheep-anti-pGFAT (MRC-PPU s343c) for immunoblotting. The secondary antibodies used were rabbit anti-mouse peroxidase (A9044, Sigma-Aldrich, Munich, Germany, 1:20,000), goat-anti-rabbit peroxidase (A9169, Sigma-Aldrich, Munich, Germany, 1:40,000), and donkey-anti-sheep peroxidase (Sigma-Aldrich). qPCR primers were obtained from Applied Biosystems, ThermoFischer: GFAT Hs00899865_m1, OGA Hs00201970_m1, OGT Hs00269228_m1, and 18S Hs03003631_g1. Gelatin from porcine skin (48720, Fluka, Bucharest, Romania) was used as a 1% solution in PBS. Thiamet G (TMG; SML0244, Sigma-Aldrich, Germany) treatment was given at 10 µM for 24 h.
Ab2739
Manufactured by Abcam
Sourced in United Kingdom
Ab2739 is a primary antibody that recognizes the GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) protein. GAPDH is a well-known housekeeping gene that is commonly used as a loading control in various experimental techniques.
Automatically generated - may contain errors
Lab products found in correlation
Ab96718, by Abcam (7 mentions)
A9044, by Merck Group (2 mentions)
A9169, by Merck Group (2 mentions)
T6557, by Merck Group (2 mentions)
Sc 74403, by Santa Cruz Biotechnology (2 mentions)
Ecltm western blotting detection reagent, by Bio-Rad (2 mentions)
Ab124807, by Abcam (2 mentions)
Ab49900, by Abcam (2 mentions)
Thiamet g, by Merck Group (2 mentions)
Ab8226, by Abcam (2 mentions)
Phosstop, by Merck Group (2 mentions)
Complete protease inhibitor cocktail, by Merck Group (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Sab4200267, by Merck Group (1 mentions)
O 6264, by Merck Group (1 mentions)
Ab4114, by Abcam (1 mentions)
Ar1178, by Boster Bio (1 mentions)
Ab77115, by Abcam (1 mentions)
Ab3340, by Abcam (1 mentions)
Ab99532, by Abcam (1 mentions)
28 protocols using ab2739
1
Antibody Characterization for Retinal Protein Analysis
2
Western Blot Analysis of Cellular Proteins
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A2780 and SKOV3 cells were lysed in protease inhibitor (AR1178, Boster, Wuhan, China) supplemented with RIPA buffer (P0013B, Beyotime, Jiangsu, China). The western blotting of the samples was performed as described previously22 (link). Briefly, the proteins were separated by SDA-PAGE and transferred to PVDF membranes. These were overnight-incubated with primary antibodies at the appropriate concentration and 4 °C. The membranes were washed with PBST (phosphate-buffered saline Tween-20). Incubation with horseradish peroxidase-conjugated secondary antibodies was performed for 1 h at room temperature (RT). The protein bands were visualized using the ECLTM Western Blotting Detection Reagent (Bio-Rad, California, USA). The antibodies used in this study were as follows: OGT (1:1000, ab96718, Abcam, USA), O-GlcNAcylation (1:1000, ab2739, Abcam, USA), SNAP-23 (1:500, ab4114, Abcam, USA), ALIX (1:1000, ab27345, Abcam, USA), and Actin (1:1 000, bs-0061, Bioss, Beijing, China).
3
Adipocyte Lipid Metabolism Regulation
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Antibodies against OGT (ab96718), OGA (MGEA5, ab124807), O-GlcNAc (RL2, ab2739), PLIN1 (ab3526), SNAP23 (ab3340), ATGL (ab99532), DGAT1 (11561-1-AP), Fsp27 (CIDE C, ab77115), and p-Ser (phosphoserine, PSR-45) (Abcam); p492-phosphorylated PLIN1 (4855) and p517-phosphorylated PLIN1 (4856) (Vala Sciences); HA (H3663) (Sigma-Aldrich); CGI-58 (ABHD5, 12201-1-AP), PLIN2 (15294-1-AP), and PLIN3 (TIP47, 10694-1-AP) (Proteintech); p563-HSL (4139), phospho-Akt (Ser473, 9271), and Akt (9272) (Cell Signaling Technology); Myc (sc-40), DGAT2 (sc-66859), and β-actin (sc-8432) (Santa Cruz Biotechnology) were purchased from the indicated sources. Horseradish peroxidase-conjugated secondary antibodies were from Santa Cruz Biotechnology. Alexa Fluor 594-conjugated secondary antibodies, BODIPY 493/503, and BODIPY 558/568 C12 fatty acid were obtained from Thermo Fisher Scientific. 4′,6-Diamidino-2-phenylindole (DAPI), OA, Fsk, and IBMX were from Sigma-Aldrich. CL-316,243 was from R&D Systems. OGT inhibitor ST045849 (ST04) was purchased from TimTec. TMG was from Cayman Chemical.
4
Western Blotting for Autophagy Proteins
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Western blotting was performed as previously described 29 (link). Briefly, after protein extraction and denaturation, an appropriate SDS-PAGE protocol (15% SDS-PAGE for VAMP8; 12% SDS-PAGE for LC3, SNAP-29 and Stx17; 10% SDS-PAGE for O-GlcNAc and p62; 8% SDS-PAGE for OGT) was selected according to the molecular weight of the protein. The primary antibody was incubated at the appropriate concentration overnight at 4 °C. The membranes were then incubated with secondary antibodies conjugated with horseradish peroxidase for 2 h at room temperature and visualized using ECLTM Western Blotting Detection Reagent (Bio-Rad, USA). The antibodies used in this study were as follows: OGT (1:1,000, Abcam, ab96718), LC3 (1:1,000, CST, 2775s), p62 (1:1,000, Abcam, ab56416), O-GlcNAcylation (1:1,000, Abcam, ab2739), SNAP-29 (1:1,000, Abcam, ab138500), Stx17 (1:1,000, MBL, PM045), VAMP8 (1:5,000, Abcam, ab76021), Actin (1:2,000, CWBIO, CW0096A).
5
YAP Protein O-GlcNAcylation Assay
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Reaction mixtures containing 1 μM purified human YAP protein (Abnova, Taiwan, # H00000116-P01), 0.125 μM human OGT (Abnova, H00008473-P01) and 10 μM UDP-GlcNAc (Sigma) in a buffer of 50 mM Tris/HCl (pH 7.5) and 1 mM DTT were incubated at 37 °C for 90 min. Then, co-IP was performed with anti-YAP antibodies (Abcam, #ab52771, 1:100) and detected by WB using anti-YAP (Santa Cruz, #sc-101199, 1:1000) and anti-O-GlcNAc (Abcam, #ab2739, 1:1000) antibodies.
6
Western Blot Analysis of O-GlcNAcylation
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Cells were prepared for western blotting in the following manner, and all the steps were conducted at 4°C. Cells, treated with inhibitors or DMSO in fresh media, (changed 3 h before treatment) were washed once with cold PBS, collected in cold PBS, centrifuged and lysed in RIPA buffer (25 mM Tris, pH 8.0, 1% NP40, 0.5% DOC, 0.1% SDS, 150 mM NaCl) supplemented with cOmplete™ protease inhibitor cocktail (Sigma), PhosSTOP™ (Sigma), and 50 μM thiamet-G (Sigma). After this, samples were loaded on an SDS-PAGE gel and transferred to nitrocellulose membrane for immunoblotting. Antibodies used in this study include: anti-OGT (24083S, CST), anti-O-GlcNAc (RL2, ab2739, Abcam) and anti-actin (ab49900, Abcam).
7
Assessing O-GlcNAcylation in HEK293T and COS-7 Cells
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To assess the degree of O-GlcNAcylation upon OGT and OGA expression in HEK293T/17 cells and COS-7 cells, the constructs were expressed in these cell lines for 2 d (Fig. S1, A and B ). The cells were lysed, and proteins were separated by SDS-PAGE and transferred onto a nitrocellulose membrane for Western blotting with an antibody against O-GlcNAcylation [RL2] (ab2739; Abcam). O-GlcNAcylation was quantified by the ratio of RL2 immunoreactivity in the entire lane to an anti-GAPDH band and then normalized to lysates from cells expressing GFP alone.
8
Quantitative Immunofluorescence Staining of Pancreatic Cancer
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For cell staining, cells grown on coverslips were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in TBS (50 mM Tris–HCl, pH 7.4, 150 mM NaCl) buffer. For specimen staining, formalin-fixed, paraffin-embedded sections of pancreatic cancer tissues were obtained. After blocking with 5% BSA (Yeasen, Shanghai) in PBS, antibodies against O-GlcNAc (Abcam, Inc., ab2739) or CA19-9 (Abcam, Inc., ab3982) at a dilution of 1/200 in 5% BSA were used to detect their expression at 4 °C overnight. After washing, the samples were stained with AlexaFluor® 488 goat anti-rabbit secondary antibody (Jackson ImmunoResearch, Inc.) or AlexaFluor® 594 goat anti-mouse secondary antibody (Jackson ImmunoResearch, Inc.) at a 1/200 dilution for 1 h at room temperature. DAPI (Southern Biotech, Inc.) was used as the nuclear counterstain. Fluorescent images were acquired using a Leica confocal laser scanning microscope (Leica, Inc.). Confocal imaging was performed in seven random fields. Mean fluorescence intensity (MFI) was calculated using Image-Pro plus software (version 6.0, Media Cybernetics, Inc.).
9
Immunofluorescence Analysis of O-GlcNAc Proteins in Oocytes
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The oocytes were fixed in 1% paraformaldehyde and 0.2% Triton X-100 in PBS for 1 h at room temperature. After 1 h, the oocytes were transferred into 3% bovine serum albumin (BSA) in PBS to be blocked for another 1 h. Next, the oocytes were incubated with fluorescent-labeled antibodies against OGA (1:200), NAT10 (1:200), O-GlcNAc Transferase (OGT) (1:200), and/or another first antibody RL2 (1:200, Abcam, ab2739) at 4°C overnight. After three washes with 0.3% BSA, oocytes incubated with RL2 first antibody were then incubated with Cy3-conjugated secondary antibody (1:500, Earthox, E031620) at room temperature for 1 h in the dark condition. The oocytes were then washed with 0.3% BSA for three times. In addition, images were taken under the inverted phase contrast confocal microscope (LSM 880, Zeiss, JENA, Germany).
10
Quantifying O-GlcNAcylated Proteins via Immunoblot
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The amount of O‐GlcNAcylated proteins was analysed by immunoblot and immunostaining. To this purpose, cells were incubated with dichloroacetate for 3 days, harvested in 2× Laemmli buffer, and proteins were separated by an 8% SDS‐PAGE. After semi‐dry transfer, the membrane was incubated with 1:1,000 mouse anti‐O‐GlcNAc antibody (Abcam Cat# ab2739, RRID:AB_303264, Cambridge, UK) in PBS followed by 1:1,000 goat anti‐mouse AlexaFluor488 (A‐11029; ThermoFischer) staining. Quantified protein content was normalized to tubulin concentration. The same antibody was used for immunostaining: The cells were grown on glass slides and fixed with 4% formalin. Next, the cells were permeabilized and blocked by 10% FBS, 0.3‐M glycerine, 1% BSA, and 0.1% Triton X‐100 in PBS. The cells were then incubated with the O‐GlcNAc antibody and Hoechst. O‐GlcNAc staining was analysed by Fiji and normalized to nuclei count. The O‐GlcNAc transferase inhibitor alloxan (10 mM, Sigma) was used as negative control (Konrad et al., 2002 ).
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