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7 protocols using ab194357

1

Protein Expression Analysis in Cells

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Total protein was prepared in RIPA lysis buffer (Beyotime, Nantong, China) on ice, then extracted by 12% SDS-PAGE and shifted to PVDF membranes (Millipore, Billerica, MA). Membranes were sealed for 1 h with 5% skim milk and incubated with primary antibodies including anti-GAPDH (ab9485, abcam), anti-Bax (ab32503, abcam), anti-Bcl-2 (ab32124, abcam), anti-caspase 3 (ab3235, abcam), anti-cleaved caspase 3 (ab2302, abcam), anti-CDCA4 (ab227953, abcam), anti-ki-67 (ab270650, abcam), anti-PCNA (ab29, abcam), and anti-EGR1 (ab194357, abcam) respectively all night at 4 °C. After being washed in TBST, secondary antibodies tagged with HRP were added and incubated for 1 h at 37 °C. The protein bands were quantified by ECL Prime Western Blotting Detection reagent (GE Healthcare, Chicago, IL).
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2

Western Blot Analysis of EMT Markers

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RIPA lysis buffer (P0013B, Beyotime, China) was used to extract proteins from cells. BCA Protein Assay Kit (P0012, Beyotime, China) was used to measure the concentration of proteins. Each cell protein lysates were mixed with 5 × SDS buffer and boiled at 100 ℃ for 10 min before western blotting. The proteins were separated in sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to the polyvinylidene difluoride (PVDF) membrane. Then, the PVDF membrane was blocked in 5% milk in tris buffered saline with 0.05% Tween 20 (TBST) for 90 min. The membranes were then incubated with primary antibodies at 4 ℃ overnight. The membranes were washed by TBST and incubated with second antibodies for 60 min and washed by TBST again. The proteins on the PVDF membrane were then visualized by an enhanced chemiluminescence detection kit (Beyotime, China) and an automatic chemiluminescence image analysis system (Tanon, China). The protein bands were further quantified by Image J software. The primary antibodies used in the study were: anti-E-cadherin (20874-1-AP, Proteintech, China), anti-N-cadherin (ab18203, Abcam, USA), anti-vimentin (VIM) (10366-1-AP, Proteintech, China), anti-SNAI2 (12129-1-AP, Proteintech, China), anti-β-actin (A1978, MilliporeSigma, USA), anti-EGR1 (ab194357, Abcam, USA), anti-KAT3B/p300 (ab275378, Abcam, USA), anti-CREBBP (CBP) (ab253202, Abcam, USA).
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3

Western Blot Analysis of Egr-1 Protein

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Cells were collected and lysed in RIPA lysis buffer (Thermo Fisher, Waltham, USA). Fifteen percent SDS-PAGE was used to separate the proteins. Subsequently, the isolated proteins were transferred to the PVDF membranes (Thermo Fisher, Waltham, USA) by semi-dry transfer. BSA solution (5–10%) was added and hatched for 1–2 h. Then, the membranes were incubated with primary antibodies against Egr-1 (#ab194357, Abcam, USA) or β-actin (#ab8226, Abcam, USA) at 25°C for 2 h. After being washed, horseradish peroxidase (HRP)-conjugated secondary antibody (1:3000, Abcam) was used to incubate with the membranes at 25 °C. One to two hours later, blots were incubated with the ECL reagents (Amersham, Little Chalfont, UK) and exposed under Amersham Imager 600 (GE, Boston, USA).
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4

Chromatin Immunoprecipitation Protocol

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Pierce™ Magnetic ChIP Kit (26157, Thermo Scientific™, Waltham, Massachusetts, USA) was used in this experiment. The cells were cultivated in 10 cm dishes and collected after being fixed with formaldehyde. Then the cell membrane and cytosol were lysed and the nucleic acids were digested by MNase. The chromatins with proteins were further obtained by sonication. Anti-IgG and anti-EGR1 (ab194357, Abcam, USA) were used as the antibody to incubate with chromatins. Protein A/G magnetic beads were used to purify the DNA. The results were further analyzed by qRT-PCR.
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5

Crosslink IP for EGR1 and p300 Detection

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Pierce Crosslink Magnetic IP/Co-IP Kit (88805, Thermo Scientific, Waltham, Massachusetts, USA) was used in this experiment. In brief, protein A/G magnetic beads were bound and crosslinked by DSS to the first antibody. Then, the cell lysates were incubated with the beads overnight. Finally, the bound antigens were eluted and subsequently analyzed by Western blotting. Anti-EGR1 (ab194357, Abcam, USA) and anti-KAT3B/p300 (ab275378, Abcam, USA) were used as the first antibody.
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6

Comprehensive Protein Expression Profiling

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For this analysis, 293T, HeLa, and HepG2 cells were grown and cultured in 6-well plates. At 90% confluence, cells were harvested and analysed with 250 μL 1× SDS loading buffer. After incubating for 10 min at 100 °C, Western blot assays were performed using 20 μL cell lysate and the antibodies against the factors, including EGR1 (Ab194357, Abcam, UK), HA(H9658; Sigma-Aldrich, USA), GAPDH (RAB0101, Frdbrio, Wuhan, China), BRF1 (SC-81405, Santa Cruz Biotech, USA.), GTF3C2 (SC-81406, Santa Cruz Biotech, USA), c-MYC (SC-40, Santa Cruz Biotech, USA), MDM2 (SC-965, Santa Cruz Biotech, USA), p53 (SC-126, Santa Cruz Biotech, USA), RhoA (SC-418, Santa Cruz Biotech, USA), PTEN (SC-7974, Santa Cruz Biotech, USA), and EGFP (TAG0070, FrdBio, Wuhan China).
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7

Immunohistochemistry for EGR1 in Lung Tissue

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We performed immunohistochemistry using ImmPRESS HRP Horse Anti-Rabbit IgG PLUS Polymer Kit (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer’s instructions. We deparaffinized the paraffin-embedded slides in xylene and rehydrated them in a graded series of ethanol. We performed antigen retrieval by autoclaving the slides in 10 mM citrate buffer, at pH 6.0. We blocked the activity of the endogenous peroxidase with 3% hydrogen peroxide for 30 min. After blocking, we incubated the slides with a 1:200 dilution of rabbit monoclonal anti–EGR1 antibody (ab194357, Abcam, Cambridge, UK) at 4°C overnight in a humidified chamber. Next, we incubated the slides with ImmPRESS HRP horse anti-rabbit IgG polymer reagent (Vector Laboratories) for 30 min, followed by 3,3′-diaminobenzidine (DAB) staining for 40 s. We counterstained the slides with Mayer hematoxylin, dehydrated in a graded series of ethanol followed by xylene, and mounted with Mount Quick (Daido Sangyo, Saitama, Japan). Paraffin-embedded normal lung sections were purchased from Novus Biologicals (62-years old, male, NBP2-30182).
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