Mouse anti gapdh

Manufactured by Cell Signaling Technology

Sourced in United States

Mouse anti-GAPDH is a primary antibody that specifically binds to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein, which is a commonly used endogenous control in various cell and molecular biology applications.

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Close spelling variants of this product

Mouse anti-GAPDH antibody Mouse monoclonal anti-Gapdh antibody Mouse monoclonal anti-GAPDH Mouse polyclonal anti-GAPDH Mouse anti-human GAPDH Rabbit anti-mouse GAPDH antibody Rabbit anti-mouse GAPDH Mouse anti-human GAPDH monoclonal antibody Mouse anti-human GAPDH antibody GAPDH mouse

46 protocols using mouse anti gapdh

Penumbra Inflammatory Pathway Analysis

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Penumbra was dissected from brain ischemia and then homogenized in the RIPA lysis buffer (Beyotime, Nantong, China) containing a whole proteinase inhibitor cocktail. A BCA protein assay kit (Beyotime) was used to determine the protein concentration. The extracted proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electrically transferred to polyvinylidene difluoride membranes. Then, the membranes were blocked with 5% nonfat milk for 1 h at room temperature. The following primary antibodies were used: mouse anti-NLRP3 (1:1,000; Adipogen, San Diego, California, USA), rabbit anti-pro–caspase-1 (1:1,000; Santa Cruz, California, USA), rabbit anti-cleaved–caspase-1 (1:3,000; Adipogen, San Diego, California, USA), goat anti–Iba-1 (1:2,000; Abcam, Cambridge, London, UK), and mouse anti-GAPDH (1:1,000; Cell Signaling Technology, Boston, Massachusetts, USA). The membranes were shaken at 60 rpm at 4°C overnight and incubated with a secondary anti-rabbit or mouse antibodies (1:10,000; Thermo Scientific, Massachusetts, USA) for 2 h at room temperature. The protein bands were visualized using Bio-Rad system.

Wang S., Wang J., Wei H., Gu T., Wang J., Wu Z, & Yang Q. (2020). Genistein Attenuates Acute Cerebral Ischemic Damage by Inhibiting the NLRP3 Inflammasome in Reproductively Senescent Mice. Frontiers in Aging Neuroscience, 12, 153.

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Western Blot Analysis of Plantaris Muscle

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Plantaris muscles were lysed in RIPA buffer (Sigma) and proteins were separated through denaturating SDS-PAGE electrophoresis using Mini-Protean TGX precast gels 4-15% (Biorad) and transferred on PVDF or Nitrocellulose (0.2μm, Biorad) membrane using the Trans-Blot turbo transfer system (Biorad). Membranes were blocked with 5% skinned milk in TBS-1% Tween (TBST) 1hr at room temperature and probed overnight at 4°C with primary antibodies in TBST 5% BSA. The following antibodies were used: mouse anti-RhoA (Santa Cruz, sc-418, 1/150), rabbit anti-P-Akt (Cell Signaling, #9271S, 1/1000), rabbit anti-Col1 (Abcam, ab34710, 1/1000), rabbit anti-Fibronectin (Sigma F3648, 1/1000), rabbit anti-Erk2 (Cell Signaling, #9102, 1/1000), rabbit anti-P-Erk1/2 (Cell Signaling, #9101, 1/1000), rat anti-TenascinC (Invitrogen, MA1-26778, 1/200), and mouse anti-Gapdh (Cell Signaling, D16H11, 1/1000). Following washing in TBST, membranes were hybridized with secondary antibodies goat anti-mouse- or goat anti-rabbit-coupled to HRP (ThermoFisher, 62–6520 and A27036, 1/10,000). Proteins were revealed using SuperSignal West Femto substrate (ThermoFisher).

Noviello C., Kobon K., Delivry L., Guilbert T., Britto F., Julienne F., Maire P., Randrianarison-Huetz V, & Sotiropoulos A. (2021). RhoA within myofibers controls satellite cell microenvironment to allow hypertrophic growth. iScience, 25(1), 103616.

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Quantitative Analysis of Cellular Proteins

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Total cellular proteins were isolated from cultured cells using lysis buffer and assayed by Western blot according to previously described procedures.15 Proteins were detected according to the manufacturer's instructions. Briefly, equal amounts of protein were denatured, electrophoresed (SDS‐PAGE gel kit, Beyotime), and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were then incubated overnight with primary antibody, washed with tris‐buffered saline plus tween 20, and then incubated with a second antibody. The proteins were detected using ECL chemiluminescence (Millipore). The primary antibodies were mouse anti‐TfR (1:1000, M‐A712, BD Pharmingen), and mouse anti‐KRAS (1:1000) and mouse anti‐GAPDH (1:1000; Cell Signaling Technology, Danvers, MA, USA). The secondary antibody was horseradish peroxidase‐conjugated rabbit anti‐mouse IgG (1:10 000; Cell Signaling Technology).

Wu Y., Xu J., Chen J., Zou M., Rusidanmu A, & Yang R. (2017). Blocking transferrin receptor inhibits the growth of lung adenocarcinoma cells in vitro. Thoracic Cancer, 9(2), 253-261.

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Western Blot Analysis of ErbB4 in Spinal Cord

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Western blot was performed as previously reported (Dong et al., 2020 (link)). Lumbar enlargement segments of the spinal cord were dissected and the dorsal and ventral horns were carefully separated under a stereo microscope. Tissues were homogenized by sonication in RIPA buffer containing protease inhibitors. Debris was cleared by centrifugation at 12,000 g for 10 min at 4°C. Samples were resolved on SDS-PAGE and transferred to nitrocellulose membranes, which were incubated in PBS (phosphate buffered saline) containing 0.1% Tween-20 and 5% BSA for 1 hr at room temperature (RT) before overnight primary antibody incubation at 4°C. After wash, membranes were incubated with HRP-conjugated secondary antibody in PBS for 1 hr at RT. After incubating with enhanced chemiluminescence, immunoreactive bands were visualized by LI-COR Odyssey infrared imaging system. Intensity of immunoreactive bands were quantitated with ImageJ (NIH), with β-actin or GAPDH as loading control. The following primary antibodies were used: rabbit anti-ErbB4 (#0618, 1:2,000, generously provided by Dr. Cary Lai) (Zhu et al., 1995 (link)), rabbit anti-p-ErbB4 (#4757, 1:200, Cell Signaling Technology), mouse anti-β-actin (#3700, 1:5,000, Cell Signaling Technology), and mouse anti-GAPDH (#97166 1:5,000, Cell Signaling Technology).

Wang H., Chen W., Dong Z., Xing G., Cui W., Yao L., Zou W.J., Robinson H.L., Bian Y., Liu Z., Zhao K., Luo B., Gao N., Zhang H., Ren X., Yu Z., Meixiong J., Xiong W.C, & Mei L. (2022). A novel spinal neuron connection for heat sensation. Neuron, 110(14), 2315-2333.e6.

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Western Blot Analysis of Protein Expression

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Tissue and cells were frozen in liquid nitrogen and homogenized in cell lysis buffer (20 mM HEPES-NaOH (pH 7.4), 25 mM KCl, 250 mM sucrose, 2 mM MgCl₂, 0.5 mM DTT and 1% digitonin (Sigma)) containing 1x Complete Protease Inhibitor and 1x PhosStop (Roche) using the Qiagen TissueLyser II. Tissue lysates were cleared by centrifugation, separated by SDS-PAGE, and transferred to nitrocellulose membranes. Membranes were blocked with 5% skim milk or 5% BSA in 1xTBS containing 0.1% Tween-20 for 1h at RT and incubated overnight with one of the following primary antibodies: rabbit anti-eGFP (GTX26556, 1:5000 in 5% milk in TBS-T), mouse anti-Lc (MA5-11860, 1:1000 in 5% milk in TBS-T), mouse-anti-GAPDH (sc-25778, 1:1000 in 5% milk in TBS-T), rabbit anti-CHC (Cell Signaling, 1:1000 in 5% BSA in TBS-T) or beta-Tubulin (Cell Signaling, 1:2000 in 5% BSA in TBS-T) followed by subsequent incubation with an HRP-conjugated secondary goat anti-rabbit or goat anti-mouse antibody (1:3000 in 5% milk or 5% BSA in TBS-T). Membranes were visualized using Clarity^™ Western ECL substrate (Biorad) and ChemiDoc^™ MP Imaging System (Biorad).

Grimm E., van der Hoeven F., Sardella D., Willig K.I., Engel U., Veits N., Engel R., Cavalcanti-Adam E.A., Bestvater F., Bordoni L., Jennemann R., Schönig K., Schiessl I.M, & Sandhoff R. (2022). A Clathrin light chain A reporter mouse for in vivo imaging of endocytosis. PLoS ONE, 17(9), e0273660.

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Western Blot Analysis of Protein Targets

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Cells were lysed with radio immunoprecipitation assay buffer (RIPA, Beyotime, China) supplemented with PMSF (Sigma), and the protein concentration was determined using the bicinchoninic acid (BCA) protein assay (Beyotime, China). Protein samples (20 µg) were mixed with Bolt LDS sample buffer (Invitrogen) and incubated for 10 min at 95 °C. Samples were run on TGX FastCast acrylamide gels (Bio-Rad, USA) and electrophoretically transferred to PVDF membranes (Millipore, USA). Membranes were incubated at 4 °C overnight with primary antibody, and then with secondary antibody for 2 h at room temperature. Primary antibodies used in the study were rabbit anti-TFRC (1:1000, ProteinTech, Wuhan, China) and mouse anti-GAPDH (1:1000, Cell Signaling). Finally, the blots were then incubated with chemiluminescence substrate, and then visualized using Bio-Rad chemiluminescence imaging system.

Luan J.C., Zeng T.Y., Zhang Q.J., Xia D.R., Cong R., Yao L.Y., Song L.B., Zhou X., Zhou X., Chen X., Xia J.D, & Song N.H. (2021). A novel signature constructed by ferroptosis-associated genes (FAGs) for the prediction of prognosis in bladder urothelial carcinoma (BLCA) and associated with immune infiltration. Cancer Cell International, 21, 414.

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Ghrelin Signaling Pathway Regulation

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Rapamycin and DMSO were obtained from Sigma-Aldrich (St Louis, MO, USA). Ghrelin peptide was purchased from Phoenix Pharmaceuticals Inc. (Burlingame, CA). Rabbit anti-phospho-mTOR (ser235/236), rabbit anti-mTOR, rabbit anti-phospho-S6 (ser235/236), rabbit anti-S6, rabbit anti-phospho-AKT (ser473), rabbit anti-AKT, rabbit anti-insulin, rabbit anti-phospho-Smad2 (ser465/467), mouse anti-Smad2, rabbit anti-phospho-Smad3 (ser423/425), mouse anti-GAPDH, and mouse anti-β-actin antibodies were obtained from Cell Signaling Technology (Beverly, MA). Rabbit anti-αSMA and mouse anti-ghrelin antibodies were purchased from Abcam (Cambridge, UK). IRDye-conjugated affinity purified anti-rabbit, anti-mouse IgGs were purchased from Rockland (Gilbertsville, PA). Goat anti-rabbit fluoresceinisothiocyanate-conjugated IgG, goat anti-mouse Texas Red-conjugated IgG, and goat anti-insulin A (C-12) antibody were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Trizol reagent and the reverse transcription (RT) system were from Promega Inc. (Madison, WI).

Yu R., Li Z., Liu S., Huwatibieke B., Li Y., Yin Y, & Zhang W. (2018). Activation of mTORC1 signaling in gastric X/A-like cells induces spontaneous pancreatic fibrosis and derangement of glucose metabolism by reducing ghrelin production. EBioMedicine, 36, 304-315.

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Immunoblotting of Signaling Pathways

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THP1 cells were lyzed in 1X SDS-PAGE loading buffer with protease and phosphatase inhibitor cocktails (Roche). Samples were resolved on a 4%−20% gradient SDS-PAGE gel. Following transfer of the proteins, the nitrocellulose membrane was blocked in 5% milk for 1h and probed with the following antibodies overnight at 4°C: goat anti-IFIT1 (Santa Cruz, 82946), Rabbit anti-phos-pho-p38 (Cell Signaling, 4511S), Rabbit anti-phospho-p65 (Cell Signaling, 3033S), Rabbit anti-p105 (Santa Cruz, sc293141), Mouse anti-GAPDH (Cell Signaling, 97166), Rabbit anti-hnRNPL (Santa Cruz, sc-32317), Rabbit anti-phospho-IRF3 (Abcam, ab76493), Rabbit anti-phospho-JNK (Cell Signaling, 9255), Rabbit anti-phospho-ERK (Cell Signaling, 4370), Rabbit anti-phospho-STAT1 (Cell Signaling, 9167S). Western blots were incubated with respective HRP-conjugated secondary antibodies and visualized using ECL reagents (Pierce).

John S.P., Sun J., Carlson R.J., Cao B., Bradfield C.J., Song J., Smelkinson M, & Fraser I.D. (2018). IFIT1 Exerts Opposing Regulatory Effects on the Inflammatory and Interferon Gene Programs in LPS-Activated Human Macrophages. Cell reports, 25(1), 95-106.e6.

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Western Blot Analysis of TRPC3, Cadherin, and GAPDH

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The procedures of Western blot were carried out as previously described [15 (link)]. Protein samples (20μg) were separated in an 8% DS-PAGE gel and transferred to a nitrocellulose membrane (#66485; PALL Life Sciences) via semi-dry transfer (350 mA, 90 min). Primary antibodies: Rabbit anti-TRPC3, 1:200, #ab 51560, Abcam; rabbit anti-Pan cadherin, 1:1000, ab6505, #ab16505, Abcam; mouse anti-GAPDH, 1:20,000, #2118S, Cell Signaling Technology. Densitometry was employed to quantify proteins of interest using the gel analyzer function of ImageJ (ver. 1.46a; National Institutes of Health, USA).

Deurloo M.H., Turlova E., Chen W.L., Lin Y.W., Tam E., Tassew N.G., Wu M., Huang Y.C., Crawley J.N., Monnier P.P., Groffen A.J., Sun H.S., Osborne L.R, & Feng Z.P. (2018). Transcription Factor 2I Regulates Neuronal Development via TRPC3 in 7q11.23 Disorder Models. Molecular Neurobiology, 56(5), 3313-3325.

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Western Blot Analysis of Protein Targets

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Total cellular proteins were lysed by RIPA buffer with protease and phosphatase inhibitors (Pierce, USA). BCA kit (Beyotime, China) was used to determine the protein concentration. Samples were separated by 10% or 12% SDS-PAGE and transferred to polyvinylidene ﬂuoride (PVDF, Millipore, USA) membrane. After incubation of the primary antibodies anti-ALKBH5 antibody (1:1,000; Sigma, USA), anti-HIF-1α antibody (1:1,000, CST, USA), anti-HIF-2α antibody (1:1,000, CST, USA), rabbit anti-AURKB antibody (1:1,000; Abcam, USA), or mouse anti-GAPDH (1:1,000; Cell Signal Technology, USA) at 4 °C for overnight, the membranes were then incubated with horseradish peroxidase (HRP)-labeled secondary antibodies (CST, USA) for 60 min at 37 °C. After washing 3 times, a chemiluminescence system (Bio-Rad, USA) and Image Lab Software (Bio-Rad, USA) were used to detect and analyze signals.

Zhang X., Wang F., Wang Z., Yang X., Yu H., Si S., Lu J., Zhou Z., Lu Q., Wang Z, & Yang H. (2020). ALKBH5 promotes the proliferation of renal cell carcinoma by regulating AURKB expression in an m6A-dependent manner. Annals of Translational Medicine, 8(10), 646.

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