Cultured cells were lysed in RIPA buffer (150 mM NaCl, 10 mM Tris–HCl pH 7.4, 0.1% SDS, 1% DOC, 5 mM EDTA, 1% Triton X-100 and 1× EDTA-free Proteinase Inhibitor Cocktail (PIC) from Roche 11873580001) for 10 min on ice, homogenized and centrifuged at 14000g/4ºC for 10 min. For western blotting, the following commercial primary antibodies were used: anti-ß-ACTIN (Santa Cruz, sc-47778), anti-CELF1 (Santa Cruz, sc-20003), anti-PDIA6 (ThermoFisher PA3-008 or Proteintech 18233–1-AP), anti-Vinculin (Sigma V9131), anti-HA tag (Abcam ab9110), anti-GFP (Invitrogen A6455), anti-Ras (BD Biosciences 610001), anti-α-Tubulin (Sigma T9026). Anti-Stubarista and anti-CSDE1 were produced in house (24 (link),28 (link)). For chemiluminescence detection, we used Goat Anti-Rabbit IgG and Goat Anti-Mouse IgG (Biorad, 1706515 and 1721011), and for infrared (IR) detection, IRDye 800CW and IRDye 680RD (LI-COR, 92632213 and 92668072). The Gels tool from ImageJ was used for quantification.
Anti celf1
Manufactured by Santa Cruz Biotechnology
Anti-CELF1 is a monoclonal antibody that specifically recognizes the CELF1 (CUGBP, Elav-like family member 1) protein. CELF1 is an RNA-binding protein involved in the regulation of mRNA splicing, translation, and stability. The Anti-CELF1 antibody can be used for the detection and analysis of CELF1 expression in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit igg, by Bio-Rad (1 mentions)
Anti vinculin, by Merck Group (1 mentions)
Anti gfp, by Thermo Fisher Scientific (1 mentions)
Anti actin, by Santa Cruz Biotechnology (1 mentions)
Anti ras, by BD (1 mentions)
Goat anti mouse igg, by Bio-Rad (1 mentions)
Irdye 800cw, by LI COR (1 mentions)
Anti α tubulin, by Merck Group (1 mentions)
Anti ha tag, by Abcam (1 mentions)
Irdye 680rd, by LI COR (1 mentions)
Anti actin, by Merck Group (1 mentions)
Rnase cocktail, by Thermo Fisher Scientific (1 mentions)
Anti pcna, by Merck Group (1 mentions)
Hybond, by Cytiva (1 mentions)
3 protocols using anti celf1
1
Western Blotting of Cultured Cells
2
RNA-Protein Interactions: A Pull-Down Assay
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Pull-down assay was performed using in vitro transcribed biotinylated VIM, FABP7 and 5′UTR msl2 RNA probes (negative control) according to Pierce™ Magnetic RNA-Protein Pull-Down Kit protocol with some modifications. Labeled RNA was captured using 100 μl of streptavidin magnetic beads in RNA Capture Buffer for 30 min at room temperature. Beads were washed twice in 20 mM Tris (pH 7.5), once in Protein-RNA Binding Buffer and 200 μl of H9 hESCs extract was added (8 μg μl−1 of total protein). Samples were incubated for 30 min at room temperature, irradiated or not with 0.15 J cm − 2 at 254-nm UV light, washed three times with Wash Buffer and eluted after 30 min of incubation at 37 °C with alternative Elution Buffer (Tris–HCl pH 7.4 10 mM, MgCl2 1 mM, NaCl 40 mM) with 2 μl of RNAse cocktail (Ambion AM2286; RNases A and T1). RNA pull-down specificity was assessed by Western blotting using anti-CSDE1 antibody (Abcam, #96124, 1:1,000), anti-CIRBP antibody (Abcam, #94999, 1:500), anti-CELF1 (Santa Cruz, c-20003, 1:500) and anti-Actin (Sigma, #A2066, 1:1,000).
3
Western Blot Analysis of Cell Lysates
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Cell pellets (equivalent to one 10-cm Petri dish at 80-90% confluence) were lysed with RIPA buffer (50 mM Tris HCl pH7.4, 150 mM NaCl, 1% sodium deoxycholate, 1% Triton X-100; 0.1% SDS; P8340 Sigma protease inhibitor). After a 10-minute incubation at room temperature, the samples were sonicated with a micro-probe at 4 ° C, and centrifuged at 10,000 rpm for 5 minutes at 4 ° C. Proteins were separated by electrophoresis and electro-transferred (Hybond C plus, GE-Healthcare) following standard western blot procedures with anti-CELF1 (SantaCruz, 3B1, sc-20003) , anti-PCNA (Sigma-Aldrich, p8825) or anti ELAV1 (SantaCruz, sc-5261) primary antibodies, and anti-mouse secondary antibodies for imaging with the Odyssey (Li-cor) imager.
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