Cells were lysed in IP lysis buffer (Pierce/ThermoFisher Scientific) containing a protease inhibitor cocktail (Sigma-Aldrich). Total lysates were incubated with anti-FLAG (14793S; Cell Signaling Technology) or anti-IgG (ab172730; Abcam) overnight at 4 °C and then mixed with Protein A/G magnetic beads (Thermo Fisher Scientific) for 2 h at room temperature. Proteins were eluted and run on SDS-PAGE for western blot analysis. The following primary antibodies were used: anti-NONO (ab70335; Abcam), anti-N-cadherin (13116S; Cell Signaling Technology), anti-CD44 (3570S; Cell Signaling Technology), anti-BCL2 (4223S; Cell Signaling Technology), anti-BAX (50599-1-Ig; ProteinTech; Wuhan, China), anti-GPX1 (ab22604; Abcam), anti-CCN1 (14479S; Cell Signaling Technology), anti-SFPQ (15585-1-AP; ProteinTech), anti-PSPC1 (ab104238; Abcam), and anti-ZEB1 (3396S; Cell Signaling Technology).
Ab70335
Manufactured by Abcam
Sourced in United States, China, United Kingdom
Ab70335 is a laboratory equipment product from Abcam. It is a high-quality device designed for use in various scientific applications. The core function of this product is to provide a reliable and precise tool for researchers and scientists.
Automatically generated - may contain errors
Lab products found in correlation
Ab104238, by Abcam (4 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Ab22604, by Abcam (2 mentions)
Ab45766, by Abcam (2 mentions)
Ab26096, by Abcam (2 mentions)
Ab92513, by Abcam (2 mentions)
Protein a g magnetic bead, by Thermo Fisher Scientific (1 mentions)
Ip lysis buffer, by Thermo Fisher Scientific (1 mentions)
Anti bcl 2, by Cell Signaling Technology (1 mentions)
Anti n cadherin, by Cell Signaling Technology (1 mentions)
Anti cd44, by Cell Signaling Technology (1 mentions)
Ab172730, by Abcam (1 mentions)
Anti ccn1, by Cell Signaling Technology (1 mentions)
Anti flag, by Cell Signaling Technology (1 mentions)
Ab1162, by Abcam (1 mentions)
Ab184305, by Abcam (1 mentions)
Ab11825, by Abcam (1 mentions)
Ab40793, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Ab32072, by Abcam (1 mentions)
14 protocols using ab70335
1
Immunoprecipitation and Western Blot Analysis
2
Protein Expression Analysis in DEN-Treated Cells
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SMMC-7721 or MHCC97h cells were treated with DEN (4 mM) or not, lysed, and then subjected to SDS-PAGE and immunoblotting, as previously described [24 (link)]. Primary antibodies against NONO (1:500; ab70335, Abcam, USA), ACLY (1:500; ab40793, Abcam, USA), Insulin like growth factor 2 mRNA binding protein 1 (IGF2BP1) (1:500; ab184305, Abcam, USA), Splicing factor proline and glutamine rich (SFPQ) (1:500; ab11825, Abcam, USA), Flag (1:1000; ab1162, Abcam, USA), Myc (1:1000; ab32072, Abcam, USA), and GAPDH (1:1000; ab181602, Abcam, USA) were used.
3
Immunofluorescence Localization of RNA-Binding Proteins
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HEK293T/17 were prepared as described above for RNA FISH. After the cells were permeabilized, the primary antibodies, anti-hnRNP K mouse monoclonal antibody [3C2]-ChIP Grade (ab39975, Abcam) and anti-PTBP1 mouse monoclonal antibody (32-4800, Thermo Fisher Scientific), were added and hybridized overnight. Alexa Fluor 647–conjugated goat anti-mouse IgG secondary antibodies (A-21236, Thermo Fisher Scientific) were used to visualize the results. Several organelle marker antibodies were tested as follows; anti-SC35 (S4045, Sigma-Aldrich), anti-nmt55/p54nrb (NONO, ab70335, Abcam), anti-ILF3 (ab133354, Abcam), anti-EF1A (sc-21758, SantaCruz), anti-ATP5 (mitochondria, ab14748, Abcam), anti-Calnexin (endoplasmic reticulum, ab202572, Abcam) and J2 monoclonal antibody (dsRNA, 10010200, SCICONS). MALAT1 probes labeled with Atto 633 (Supplementary Table S2 ).
4
Western Blotting and Co-Immunoprecipitation
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Western blotting was performed as previously described.21 Briefly, cells were harvested and lysed using cell lysis buffer (Cell Signaling Technology, Danvers, MA). After measuring the protein concentration using BCA kit (Thermo Fisher), protein was loaded into 10% polyacrylamide sodium dodecyl sulfate gel after transfer onto polyvinylidene difluoride membranes (Millipore, Burlington, MA) and incubated with primary antibodies at 4°C overnight. polyvinylidene difluoride membranes were washed 3 times with phosphate-buffered saline (PBS) for a total of 15 minutes and incubated with specific secondary antibodies (1:10000; Cell Signaling Technology) for 1 hour at room temperature. Later the signals were detected by using electrochemiluminescence kit (Pierce). Primary antibodies against IFNGR1 (1:1000, ab134070; Abcam, Cambridge, UK), glyceraldehyde-3-phosphate dehydrogenase (1:2000, GAPDH) (ab8245; Abcam), STAT1 (1:1000, 14994; Cell Signal Technology), phosphorylated STAT1 (pSTAT1) (1:1000, 9167; Cell Signal Technology), NONO (1:1000, ab70335; Abcam), flag (1:2000, ab18230; Abcam), Phospho-(Ser/Thr) (1:500, ab117253; Abcam), and Phospho-Tyrosine (P-Tyr) (1:500, 8954; Cell Signal Technology) were used. Co-immunoprecipitation was performed as previously described.5
5
Cell Lysis and Western Blot Analysis
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Cells were lysed with RIPA buffer (50 mM Tris–HCl, 150 mM NaCl, 5 mM EDTA, and 0.5% Nonidet P-40) supplied with a protease inhibitor cocktail (Millipore, 539131-1VL) on ice for 30 min and were then centrifuged at 14,000 rpm for 30 min at 4 °C. Supernatants were added to a 1/5 volume of 5× SDS loading buffer, boiled for 10 min, separated by SDS–PAGE, and transferred to polyvinylidene fluoride (PVDF) membranes.
The membranes were incubated with primary antibodies overnight at 4 °C and with secondary antibodies for one hour at room temperature. After extensive washing, the membranes were visualized with an ECL kit (Beyotime Ltd., Shanghai, China) for protein detection. Antibodies against the Myc tag (3946 S), HA tag (3724 S), and Flag tag (2368 S) were purchased from Cell Signaling Technology (Boston, USA). The antibodies against NONO (11058-1-AP), SAMHD1 (12586-1-AP), DCAF1 (11612-1-AP), and Actin (20536-1-AP) used for western blotting were purchased from Proteintech (Wuhan, China). The antibodies against NONO (ab70335) and SAMHD1 (ab264335) used for co-IP were obtained from Abcam (Cambridge, UK).
The membranes were incubated with primary antibodies overnight at 4 °C and with secondary antibodies for one hour at room temperature. After extensive washing, the membranes were visualized with an ECL kit (Beyotime Ltd., Shanghai, China) for protein detection. Antibodies against the Myc tag (3946 S), HA tag (3724 S), and Flag tag (2368 S) were purchased from Cell Signaling Technology (Boston, USA). The antibodies against NONO (11058-1-AP), SAMHD1 (12586-1-AP), DCAF1 (11612-1-AP), and Actin (20536-1-AP) used for western blotting were purchased from Proteintech (Wuhan, China). The antibodies against NONO (ab70335) and SAMHD1 (ab264335) used for co-IP were obtained from Abcam (Cambridge, UK).
6
Western Blot Analysis of Protein Expression
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The cells were harvested and lysed using total protein lysis buffer (Cell Signaling Technology, Inc., Danvers, MA, USA). A total of 30 µg of protein was separated on a 10% polyacrylamide-SDS gel, blotted onto a PVDF membrane (Millipore, Billerica, MA, USA) and blocked with 5% non-fat milk (Sangon Biotech Co., Ltd., Shanghai, China). After incubation with the primary antibody overnight at 4°C and the corresponding secondary antibody at 37°C for 1 h, the membrane was developed using an ECL kit (Pierce, Rockford, IL, USA). The antibodies used were as follows: Rabbit anti-GAPDH (1:1,000) (GP10353; Nuoyang, Hangzhou, China); rabbit anti-p-p65 (1:1,000; no. 3033); rabbit anti-p-p38 (1:1,000; no. 4511); rabbit anti-p-JNK (1:1,000; no. 9255); rabbit anti-p-ERK (1:1,000; no. 3510) (all from Cell Signaling Technology, Inc.); rabbit anti-cluster of differentiation 36 (CD36; 1:1,000; ab133625); rabbit anti-LOX-1 (1:1,000; ab60178); rabbit anti-NONO (1:1,000; ab70335); rabbit anti-SFPQ (1:1,000; ab38148) (all from Abcam, Shanghai, China); goat anti-rabbit (1:5,000; GP853); and goat anti-mouse (1:5,000; GP843) (both from Nuoyang).
7
Immunohistochemical Analysis of NONO, Ki67, and GPX1
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Sections (5 μm) were cut from paraffin-embedded tissues and incubated with primary antibody. Detection was performed through incubation with the species appropriate secondary antibody conjugated to horse radish peroxidase and the substrate DAB. The following primary antibodies were used: rabbit anti-NONO (ab70335, 1:200; Abcam; Waltham, MA, USA), rabbit anti-Ki67 (GB13030-2, 1:1,000; Servicebio; Wuhan, China) and rabbit anti-GPX1 (ab22604, 1:200; Abcam). Staining was evaluated independently to determine the histological score according to the proportion of positive staining cells and staining intensity.
8
Protein Expression Analysis by Western Blot
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Cells were lysed in ice-cold whole cell extract buffer B [50 mM Tris–HCl (pH 8.0), 4 M urea, and 1% Triton X-100] supplemented with complete protease inhibitor cocktail (Roche). The cell extracts were resolved with SDS-PAGE and analyzed with western blotting. The protein bands were visualized with ECL Blotting Detection Reagents. The antibodies used for western blotting included an anti-P54nrb antibody (Abcam, ab70335), anti-PSPC1 antibody (Abcam, ab104238), anti-SFPQ antibody (Abcam, ab11825), anti-STAT3 antibody (CST, 124H6), anti-pSTAT3Y705 antibody (Abcam, ab76315), anti-HSV-1 ICP0 antibody (Abcam, ab6513), anti-HSV-1 TK antibody (Santa Cruz Biotechnology, sc-28037), and anti-β-actin antibody (Proteintech, 60008-1-Ig).
9
Immunoprecipitation of SRSF2 interactors
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The immunoprecipitation assay was performed using the Immunoprecipitation Protein G Dynabeads® kit (Invitrogen, 10007D) according to the manufacturer’s protocol. Briefly, HSV-1 or Mock infected HeLa cell lysates were incubated with antibodies against SRSF2 (Abcam, ab11826) and after washing, bound proteins were detected by western blotting with antibodies against PSPC1 (Abcam, ab104238), P54nrb (Abcam, ab70335), P300 (Abcam, ab59240), CBP (Abcam, ab50702), SRSF2 (Santa Cruz Biotechnology, sc-10252), EZH2 (Abcam, ab186006), or beta-actin (Proteintech, 60008-1-Ig).
10
NONO and PSPC1 RIP-qPCR and GPX1 mRNA Binding
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RIP was performed using the EZ-Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Merck Millipore; Burlington, MA, USA). In brief, cells were cross-linked with 1% formaldehyde and lysed with protease and RNase inhibitors. Magnetic beads preincubated with IgG or antibody specific for NONO (ab70335; Abcam) or PSPC1 (ab104238; Abcam) were incubated with lysates at 4 °C overnight. Eluted RNAs were purified and detected with qPCR. Total RNA was regarded as the input control.
Human GPX1 pre-mRNA (sense and antisense; GenePharma) and mRNA (GenePharma) were transcribed in vitro using the Transcript Aid T7 High Yield Transcription Kit (ThermoFisher Scientific). mRNAs were 3' end labeled with biotin using the RNA 3' End Desthiobiotinylation Kit (ThermoFisher Scientific), and associated proteins were pulled down in coprecipitation assays and examined on western blot.
Human GPX1 pre-mRNA (sense and antisense; GenePharma) and mRNA (GenePharma) were transcribed in vitro using the Transcript Aid T7 High Yield Transcription Kit (ThermoFisher Scientific). mRNAs were 3' end labeled with biotin using the RNA 3' End Desthiobiotinylation Kit (ThermoFisher Scientific), and associated proteins were pulled down in coprecipitation assays and examined on western blot.
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