AtT‐20 cells that were grown to 60%–70% confluence in regular medium in 6‐well plates were incubated with bexarotene in 2% charcoal‐stripped FBS medium for 24 hours. Then, the cells were washed with cold PBS and lysed in cell lysis buffer (Beyotime, Shanghai, China) with protease inhibitor cocktail (Bimake, Shanghai, China) on ice for 10 minutes. Then, the lysates were collected and centrifuged at 12 000 g for 20 minutes. Protein concentrations were quantitated by using a BCA protein assay kit (Thermo Fisher), and 20 µg of total proteins were separated on the SDS/PAGE gels, which were then transferred to polyvinylidene difluoride membranes. Blots were probed with GAPDH (ab181602, abcam), TR4 (ab109301, abcam), POMC (ab32893, abcam), PARP (ab227244, abcam), C‐caspase‐3 (ab2302, abcam), cyclin D1 (#2978, CST), CDK1 (#9116, CST), cyclin B1 (#4138, CST), cyclin A2 (#4656, CST) primary antibodies. Horseradish peroxidase (HRP)‐linked anti‐rabbit IgG and anti‐goat IgG secondary antibodies were incubated against each primary antibody, and the hybridization signals were detected by using an enhanced chemiluminescence detection kit (Fdbio‐tech).
Ab32893
Manufactured by Abcam
Ab32893 is a primary antibody product manufactured by Abcam. It is designed to detect the target protein in biological samples using standard immunodetection methods.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab109301, by Abcam (1 mentions)
Ab227244, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Protease inhibitor cocktail, by Selleck Chemicals (1 mentions)
Cell lysis buffer, by Beyotime (1 mentions)
Ab2302, by Abcam (1 mentions)
Ab135549, by Abcam (1 mentions)
Alexa fluor 568 donkey anti goat, by Thermo Fisher Scientific (1 mentions)
Animal free blocker, by Vector Laboratories (1 mentions)
Sc 10365, by Santa Cruz Biotechnology (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Ab300071, by Abcam (1 mentions)
Ab259945, by Abcam (1 mentions)
Ab184238, by Abcam (1 mentions)
Gel pro analyzer 4, by Media Cybernetics (1 mentions)
Bca protein assay kit, by CWBIO (1 mentions)
3 protocols using ab32893
1
Bexarotene Regulates Protein Expression in AtT-20 Cells
2
Multimodal Immunofluorescence Analysis of Pituitary and Adrenal Tissues
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Murine pituitary and adrenal glands and human corticotroph adenomas and normal autopsy-derived pituitary tissues (see the Human Tissue Collection section) were fixed in 10% formalin and embedded in paraffin. After deparaffinization and antigen retrieval, slides were blocked in animal free blocker (Vector Laboratories, Burlingame, CA), incubated overnight with rabbit polyclonal anti-EGFR (4267; Cell Signaling Technology; RRID:AB_2246311), goat monoclonal anti-POMC (ab32893; Abcam; RRID:AB_777375), mouse monoclonal anti-E2F1 (ab135251; Abcam; RRID:AB_2631246), rabbit polyclonal anti-pS337-E2F1 Ab (ab135549; Abcam; RRID:AB_2631245), goat monoclonal anti-growth-hormone (anti-GH; sc-10365; Santa Cruz Biotechnology; RRID:AB_2111154), or goat monoclonal antiprolactin (anti-PRL; sc-7805; Santa Cruz Biotechnology; RRID:AB_2170961). After washing, samples were incubated with Alexa Fluor donkey anti-mouse 488 (H+L, 1:500 dilution; Invitrogen), Alexa Fluor donkey anti-goat 568 (Invitrogen), and Alexa Flour donkey anti-rabbit 647 (Invitrogen) mounted with Prolong Gold antifade reagent (Invitrogen). Confocal microscope images were obtained using True Confocal Scanner (Leica Microsystems, Buffalo Grove, IL) in a dual-emission mode to separate autofluorescence from specific staining.
3
Quantitative Protein Analysis of Neuroendocrine Tissues
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The hypothalamus, pituitary, and cavernous body tissues were individually ground, homogenized using RIPA lysis buffer. The homogenates were rested on ice for 30 min, and immediately subjected to centrifugation with 12,000 × g in 4°C for 15 min, then the supernatant was collected. The total protein concentration in supernatant was determined according to the BCA Protein Assay Kit (Cwbio). 50 µg of total protein was separated using SDS-PAGE. The electrophoresis peptides were transferred to the PVDF membranes. The PVDF membranes were then blocked for 2 h using 5% nonfat dried milk and probed with antibodies at 4°C. After washing 3 times using TBST, the PVDF membranes were incubated for 2 h using horseradish peroxidase-conjugated secondary antibodies, and then ECL (a chemiluminescence agent) was used to develop the protein bands. The signal intensity was analyzed using Gel-Pro Analyzer 4.0 software (Media Cybernetics, Silver spring, MD, United States). The GAPDH was used as an internal control to assess the protein loading equivalence. The eNOS antibodies (1:500,ab300071), PDE5A1 antibodies (1:500, ab259945), ACTH antibodies (1:500, ab32893), CRH antibodies (1:500, ab184238), and GAPDH antibodies were purchased from Abcam.
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