The plasmids with FLAG-tagged, full-length or truncated PITX2 were transfected into HEK293 cells that had been cultured overnight in 6-well plates by Fugene HD transfection reagent (Roche, IN). Transfection efficiency was monitored by green fluorescence 48 hours later to ensure the consistency of experiments. Transfected cells were then lysed by the cell lysis buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, protease inhibitors) and incubated with primary antibodies at 4°C overnight before being precipitated by protein A beads (Pierce). Immunoprecipitated samples were boiled in the SDS loading buffer and subjected to immunoblotting analysis as described previously [9 (link)]. Antibodies used for immunoprecipitation were: FLAG M2 (Sigma, MO); PTIP, RBBP5 and ASH2L (Bethyl laboratory); MLL4/KMT2D (a gift from Dr. Kai Ge, NIDDK/NIH). Antibodies used for immunoblotting were: PITX2 (Abnova); PTIP, RBBP5 and ASH2L (Bethyl laboratory); hnRNP U (Abcam).
Hnrnp u
Manufactured by Abcam
Sourced in United Kingdom
HnRNP U is a RNA-binding protein that plays a role in the regulation of pre-mRNA processing. It is involved in various cellular processes, including mRNA transport, stability, and translation.
Automatically generated - may contain errors
Lab products found in correlation
Hnrnp l, by Abcam (2 mentions)
Pitx2, by Abnova (1 mentions)
Flag m2, by Merck Group (1 mentions)
Immunoshot reagent 1, by Cosmo Bio (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
Trueblot anti rabbit igg hrp, by Rockland Immunochemicals (1 mentions)
Hnrnp a1 d21h11, by Cell Signaling Technology (1 mentions)
Nucleolin, by Abcam (1 mentions)
β actin, by Merck Group (1 mentions)
Vdac 4866, by Cell Signaling Technology (1 mentions)
Rps6 2217, by Cell Signaling Technology (1 mentions)
Hdac1, by Cell Signaling Technology (1 mentions)
Gapdh, by Merck Group (1 mentions)
Immobilon chemiluminescent hrp substrate, by Merck Group (1 mentions)
Hnrnp a2 b1, by Novus Biologicals (1 mentions)
β actin, by Thermo Fisher Scientific (1 mentions)
Hnrnpa1, by Cell Signaling Technology (1 mentions)
α tubulin, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Chemidoc system, by Bio-Rad (1 mentions)
5 protocols using hnrnp u
1
PITX2 Protein Interactome Analysis
2
Western Blotting Procedure for Protein Analysis
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Western blotting was performed as previously described69 (link). Briefly, lysate samples were separated on 5–20% gradient polyacrylamide gel by SDS–PAGE following electrical transfer to PVDF membranes (GE Healthcare). After blocking with 5% dry milk, membranes were probed with the appropriate primary antibodies diluted in Immunoshot Reagent 1 (Cosmo bio, Tokyo, Japan) overnight at 4 °C. HRP-conjugated corresponding secondary antibodies (GE Healthcare) were subsequently used. Trueblot anti-rabbit IgG HRP (Rockland, Limerick, PA, USA, 1:1,000) was used as the secondary antibody to avoid interfering immunoprecipitated immunoglobulin heavy and light chains. Bound antibodies were detected using Immunostar LD reagents (Wako). The following antibodies were used: Hnrnp-U (#ab180952, 1:1,000) and Nucleolin (#ab22758, 1:1,000) from Abcam (Cambridge, UK); HA tag (#561, 1:10,000) and Syncrip (#RN046PW, 1:1,000) from MBL; Hnrnp-A2/B1 (#R4653, 1:1,000) and β-actin (#A1978, 1:2,000) from Sigma-Aldrich; and YBX1 (D299, 1:1,000), Igf2BP1 (D33A2, 1:1,000) and HnrnpA1 (D21H11, 1:1,000) from Cell Signaling Technology (CST, Danvers, MA, USA).
3
Quantifying Protein Expression in Cellular Immunostaining
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Cells were sorted, seeded on coverslips, fixed, immunostained, and the intensity quantified as described in Iborra and Buckle (2008) (link).
InFigures 1 , 2 , and 3 , the protein antibodies used were MTOR(2983), PFKP(12746), FASN(3180), HK2(2867), LDHA(2012), PRPS6(4858) (Cell Signaling); SF3B4(AB66659), HNRNPU(AB20666), NFE2L2(AB31163), EPRS(AB31531), RRS(AB31537), HK4me3(AB8580) (Abcam); HNRNPA0(SC-16509), EP300(SC-584), TBP(SC-56794), GTF2F(SC-235), PKM2(SC-365684), UBTF(SC-13125), RNA Pol II(RPB1)(SC-9001) (Santa Cruz Biotechnologies); CLU(HPA000572), SRSF2(HPA049905), MCL1(HPA008455), TUBB(T8328), G6PD(HPA000834), PDIA2(HPA051692), GAPDH(G9545), PTBP1(WH0005725M1) (Sigma-Aldrich); GLUT1(AB1340) (Chemicon); BrU(MD5010) (Caltag); H4K20me2(39173), H3K36me2(39255), H4K16(39929), H3K27me3(61017) (Active Motifs); and PABP (kindly provided by Dr. Amelia Nieto).
ForFigure 5 B, the 24 antibodies used for protein quantification were HK2(2867), HDAC1(5356), LDHA(2012), RPS6(2217), EIF4B(13088), VDAC(4866), PFKP(12746) (Cell Signaling); G6PD(HPA000834), MCL1(HPA008455), TOMM22(HPA003037), CLU(HPA000572), PTBP1(WH0005725M1) (Sigma-Aldrich); NPM1(SC-6013), NCL(SC-8031), UBTF(SC-13125), PKM2(SC-365684), GTF2F(SC-235), BCL2(SC-492) (Santa Cruz Biotechnology); NFE2L2(AB31163), SF3B4(AB66659), IMMT(AB110329) (Abcam); GLUT1(AB1340) (Chemicon); EIF5(611976), EIF6(611120) (BD).
In
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4
Western Blot Analysis of hnRNP Proteins
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For Western analysis, proteins were resolved on Tris–Glycine gels, transferred to nitrocellulose or PVDF membranes (Millipore), and probed with the following antibodies: hnRNP A2/B1 (Novus NB120-6102), hnRNP K (Novus NBP2-24531), hnRNP A1 (Cell Signaling K350), hnRNP U (Abcam 180952), hnRNP L (Abcam 6106), β-actin (Thermo MA5-15739), EZH2 (Cell Signaling 3147), and α-tubulin (Santa Cruz sc-5286). Immobilon Chemiluminescent HRP Substrate from Millipore was used for detection using a BioRad ChemiDoc system. ImageJ was used to quantify each blot.
5
Chromatin Immunoprecipitation for Regulatory Proteins
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C33A cells were fixed with 1% formaldehyde for 10 min at room temperature and chromatin extracted as described in Ryme et al. (2009 (link)). For RNA regulatory protein 1.5–2% formaldehyde was used (Görnemann et al. 2005 (link)). The chromatin was fragmented by sonication to fragments with a mean length of 500 bp. The antibodies used: BRM, SAM68, hnRNPL, hnRNPU, BAF155/SMARCC1, BAF250/ARID1, BAF200/ARID2, and BAF180/PBRM1 were purchased from Abcam, BAF200/ARID2 and BAF180/PBRM1 were from Bethyl Laboratories Inc and BRD9 were from Cell signalling and Anova (Supplementary Table S6). Primers used in the analysis are presented in Supplementary Table S5. The standard was SD, and p value calculated according to Student’s t test.
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