Ab79237

Manufactured by Abcam

Sourced in United Kingdom, United States

Ab79237 is a laboratory equipment product. It serves as a core function for specific laboratory applications. A detailed description is not available while maintaining an unbiased and factual approach.

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Lab products found in correlation

6 protocols using ab79237

Antibody Protocol for Immunofluorescence Staining

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The mouse monoclonal antibody against ECD was previously described (31 (link), 39 (link)). The antibodies used in these studies include those against PRPF8 (ab79237), DDX39A (ab96621), LRPPRC (ab97505), and ALY (ab202894) and were from Abcam. Antibodies against CRM1 (46249) and ErbB2 (2242S) were purchased from Cell Signaling. For immunofluorescence staining, we used anti-ECD (catalog number HPA006465; Sigma-Aldrich) and anti-DDX39 (catalog no. sc-271395; Santa Cruz Biotechnology) antibodies; secondary antibodies tagged with Alexa Fluor 488 (A32731 and A32723) and Alexa Fluor 568 (A-11011 and A-11031) and anti-FLAG antibody (F3165) were from Sigma-Aldrich.

Saleem I., Mirza S., Sarkar A., Raza M., Mohapatra B., Mushtaq I., Kim J.H., Mishra N.K., Alsaleem M.A., Rakha E.A., Qiu F., Guda C., Band H, & Band V. (2021). The Mammalian Ecdysoneless Protein Interacts with RNA Helicase DDX39A To Regulate Nuclear mRNA Export. Molecular and Cellular Biology, 41(7), e00103-21.

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Immunoprecipitation of HA-tagged Proteins

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For immunoprecipitations, 2 × 10⁶ B cells per sample were lysed in triton lysis buffer supplemented with benzonase, lysolecithin, EDTA-free protease inhibitors (Roche) and PhosSTOP (Roche) and rotated for 30 min at 4°C. Clarified lysates (after 10 min centrifugation at 21 000 g) were either used directly for assessing protein abundance or were incubated 1 h at 4°C under constant mixing and addition of 25 μl anti-HA resin (Roche). The resin was washed three times with the same buffer and proteins boiled off the beads in sample buffer. To assess protein abundance 0.5 × 10⁶ B cells per sample were lysed as before. The proteins were visualized by western blotting using antibodies against HA (3F10, Roche), CDC5L (BD612362, BD Biosciences), CTNNBL1 (pab19409, Abnova), CWC15 (25293–1-AP, Proteintech), PRPF19, PRPF8, PLRG1, Lamin B1, β-tubulin and β-actin (ab27692, ab79237, ab86050, ab16048, ab6046, ab8224, from Abcam).

van Maldegem F., Maslen S., Johnson C.M., Chandra A., Ganesh K., Skehel M, & Rada C. (2015). CTNNBL1 facilitates the association of CWC15 with CDC5L and is required to maintain the abundance of the Prp19 spliceosomal complex. Nucleic Acids Research, 43(14), 7058-7069.

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Immunohistochemical Analysis of PRPF8 in HCC

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Immunohistochemical (IHC) analysis of PRPF8 was performed on a representative set of FFPE samples of paired nontumor adjacent and HCC tissue (n = 14). A monoclonal anti-PRPF8 antibody (ab79237, Abcam, Cambridge, UK) diluted 1:250 was used^{25 (link)}. Two independent pathologists performed histopathologic analysis of tumors following a blinded protocol. In this analysis, +, ++, and + ++ indicate a low, moderate, and high staining intensity, respectively.

López-Cánovas J.L., Hermán-Sánchez N., del Rio-Moreno M., Fuentes-Fayos A.C., Lara-López A., Sánchez-Frias M.E., Amado V., Ciria R., Briceño J., de la Mata M., Castaño J.P., Rodriguez-Perálvarez M., Luque R.M, & Gahete M.D. (2023). PRPF8 increases the aggressiveness of hepatocellular carcinoma by regulating FAK/AKT pathway via fibronectin 1 splicing. Experimental & Molecular Medicine, 55(1), 132-142.

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Immunostaining of Cell Culture Samples

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Cells were fixed with 4% paraformaldehyde (PFA), washed twice in PBS, and placed in blocking solution (3% normal goat or donkey serum and 0.5% Triton-X 100 in PBS) for 1 h at room temperature. Cells were then incubated overnight at 4°C with one of the following primary antibodies: anti-PRPF8 (1: 200, Santa Cruz sc55534, Abcam ab79237), rabbit anti-CRALBP (1:250, Abcam), rabbit anti-ZO1 (1:50, Invitrogen), and mouse anti-Na/K-ATPase (1:100, Santa Cruz). The following day, cells were washed three to five times in PBS and incubated with an appropriate secondary antibody (1:500, Invitrogen). After secondary antibody incubation, nuclei were stained with 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) (Life Technologies, #D1306) and washed three times in PBS. The coverslips were mounted using Vectashield Mounting Medium (Vector Lab, Burlingame, CA, United States) and imaged on Leica confocal microscope SP8.

Arzalluz-Luque Á., Cabrera J.L., Skottman H., Benguria A., Bolinches-Amorós A., Cuenca N., Lupo V., Dopazo A., Tarazona S., Delás B., Carballo M., Pascual B., Hernan I., Erceg S, & Lukovic D. (2021). Mutant PRPF8 Causes Widespread Splicing Changes in Spliceosome Components in Retinitis Pigmentosa Patient iPSC-Derived RPE Cells. Frontiers in Neuroscience, 15, 636969.

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Protein Analyses using Western Blotting

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SDS-PAGE and western blot analyses were performed using standard protocols. The following primary antibodies were used: rabbit anti-TAP (CAB1001, Thermo Scientific-Pierce), anti-Myc Tag clone 4A6 (05-724, Millipore), rabbit anti ERα (sc-543, Santa Cruz Biotechnology), mouse anti-AGO2/eIF2C2 (ab57113, Abcam), rabbit polyclonal to FXR1 (ab50841, Abcam), rabbit plyclonal to integrin beta 4 binding protein (EIF6; ab77298, Abcam), anti-PRPF8 antiboby (ab79237, Abcam), mouse monoclonal anti-AGO1 clone 4G7-E12 (MABE143, Millipore), mouse anti-β-actin (A1978, Sigma Aldrich), mouse monoclonal to Dicer (ab14601, Abcam), anti-TRBP2 (H-57; sc-292550, Santa Cruz Biotechnology).

Tarallo R., Giurato G., Bruno G., Ravo M., Rizzo F., Salvati A., Ricciardi L., Marchese G., Cordella A., Rocco T., Gigantino V., Pierri B., Cimmino G., Milanesi L., Ambrosino C., Nyman T.A., Nassa G, & Weisz A. (2017). The nuclear receptor ERβ engages AGO2 in regulation of gene transcription, RNA splicing and RISC loading. Genome Biology, 18, 189.

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Immunoblotting for Protein Expression

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Cells were harvested with Versene (8 g/l NaCl, 0.2 g/l KCl, 1.15 g/l Na2PO4, 0.2 g/l EDTA, 0.1 g/l Phenol red, pH 7.34), washed in ice-cold PBS and lysed on ice in a RIPA buffer with fresh protease inhibitors (Roche Complete, Roche Applied Science, Basel, Switzerland) and Phosphatase inhibitors (PhosSTOP, Roche Applied Science). Protein (25 μg) was electrophoresed through a 4–12% Bis-Tris gel, blotted to a PVDF membrane (iBLOT, Invitrogen) and immunoblotted using antibodies to: β-tubulin (Sigma T8328, Sigma-Aldrich, St. Louis, MO, USA), Mcl1 (Santa Cruz SC-819, Santa Cruz Biotechnology, Dallas, TX, USA), PSMD14 (Sigma HPA002114, Sigma-Aldrich), PRPF8 (Abcam ab79237, Cambridge, MA, USA), and SART1 (Abcam ab88583). Rabbit anti-Hub1(UBL5) antibody was a kind gift of Dr. Hideki Yashiroda (University of Tokyo, Tokyo, Japan). Blots were scanned, and densitometry was performed by the UN-SCAN-IT 6.1 software (Silk Scientific Inc, Orem, UT, USA).

Laetsch T.W., Liu X., Vu A., Sliozberg M., Vido M., Elci O.U., Goldsmith K.C, & Hogarty M.D. (2014). Multiple components of the spliceosome regulate Mcl1 activity in neuroblastoma. Cell Death & Disease, 5(2), e1072-.

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