Western blot experiment was performed given the previously described methods (Sun et al., 2019 (link)). CD4+ T cells, Jurkat T cells, naive CD4+ T cells and mouse spleen cells were harvested. The proteins were extracted with RIPA lysis buffer (Beyotime, Shanghai, China) and the protein concentrations were quantified using a bicinchoninic acid (BCA) Protein Assay Kit (TaKaRa). The same amount of protein samples were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE, Thermo Fisher Scientific) and then transferred into polyvinylidene fluoride (PVDF) membranes (Thermo Fisher Scientific). The membranes were blocked with 5% skimmed milk and then incubated with anti-HUWE1 (ab70161, 1:2,000, Abcam), anti-FoxP3 (ab20034, 1:1,000, Abcam), anti-Ets-1 (ab220361, 1:1,000, Abcam), anti-p-Ets-1 (44-1107G, 1:1,000, ThermoFisher Scientific), anti-MEK1 (ab32576, 1:10,000, Abcam) and anti-GAPDH (ab8245, 1:500, Abcam) overnight at 4°C. The membranes were washed with TBST and incubated with the secondary antibody (ab205718, 1:2,000, Abcam) for 1 h at room temperature. The enhanced chemiluminescence reagents (Thermo Fisher Scientific) and the Versadoc MP 4000 imaging system (Bio-Rad) were performed to visualize protein bands.
Ab32576
Manufactured by Abcam
Sourced in United States
Ab32576 is a laboratory antibody product. The core function of this product is to bind to a specific target molecule.
Automatically generated - may contain errors
Lab products found in correlation
Ab184699, by Abcam (2 mentions)
Ab92547, by Abcam (2 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Versadoc mp 4000 imaging system, by Bio-Rad (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Bca protein assay kit, by Takara Bio (1 mentions)
Sds page, by Thermo Fisher Scientific (1 mentions)
Ab220361, by Abcam (1 mentions)
Ab70161, by Abcam (1 mentions)
Ab20034, by Abcam (1 mentions)
Ab205718, by Abcam (1 mentions)
Ab8245, by Abcam (1 mentions)
Ab68153, by Abcam (1 mentions)
Ab227703, by Abcam (1 mentions)
Ab80590, by Abcam (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Abcam (1 mentions)
Enhanced chemiluminescence system, by Thermo Fisher Scientific (1 mentions)
Ab96507, by Abcam (1 mentions)
Ab245117, by Abcam (1 mentions)
Ab16700, by Abcam (1 mentions)
5 protocols using ab32576
1
Western Blot Analysis of T-cell Proteins
2
Immunohistochemical Analysis of Tumor Samples
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Tumor samples were harvested and fixed using formalin, followed by paraffin-embedment. Sections were prepared into sections (5-µm thickness) for staining protocol. Sections were first dewaxed in xylene and rehydrated with serial ethanol gradients and washed in deionized water. Subsequently, sections were blocked in PBS with 1% BSA, 1% donkey serum and 0.3% Triton X-100 and 0.01% sodium azide for 1 h, room temperature. After blocking, the slides were exposed to MEK (1:200, Abcam, ab32576), STAT3 (1:200, Abcam, ab68153), PDCD4 (1:200, Abcam, ab80590), and FAP antibody (1:200, Abcam, ab227703) at 4 °C overnight, washed and incubated in biotinylated link universal antiserum for 1 h at room temperature. Primary antibodies were diluted in blocking buffer and incubated in the cold overnight. The next day, sections were washed in PBST 3 times and incubated with secondary antibodies in blocking buffer (1 h, room temperature). Stained sections were then observed under a microscope and micrographed using a mounted digital camera.
3
Quantitative Protein Analysis Workflow
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Lysed in RIPA buffer, CC cells were subjected to protein quantification via a bicinchoninic acid assay. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis was applied for the separation of proteins, followed by transferring to polyvinylidene difluoride membranes. After blocking, the membrane was incubated with primary antibodies. Further, the horseradish peroxidase-conjugated secondary antibodies (Abcam, Cambridge, UK) were supplemented. The enhanced chemiluminescence system (Thermo Fisher Scientific) was employed for evaluating the protein bands, and the proteins were quantified via Image J software (National Institutes of Health, Bethesda, MD, USA). The primary antibodies used in this study included anti-FLOT2 (1:3000, ab96507, Abcam), Vimentin (1:1000, ab16700, Abcam), N-cadherin (1/1000, ab245117, Abcam), E-cadherin (1 µg/ml, ab231303, Abcam), MEK (1:10000, ab32576, Abcam), p-MEK (1 µg/ml, ab278716, Abcam), ERK1/2 (1/10000, ab184699 Abcam), and p-ERK1/2 (0.2 ng/ml, ab176660, Abcam).
4
Western Blot Analysis of Signaling Proteins
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The transfected cells were collected after 24 h, and the cells were lysed using RIPA lysis buffer in order to extract total protein. The protein concentration was determined using the BCA method. Proteins were then separated by 10% SDS-PAGE gel electrophoresis, and transferred to a membrane, blocked with 5% skimmed milk powder at room temperature for 1 h. The membranes were then incubated with primary antibodies targeting Pax4 (PA1-108, ThermoFisher), Grb2 (ab32111; Abcam), p-Raf (ab157201; Abcam), T-Raf (ab230850; Abcam), p-MEK (ab278562; Abcam), t-MEK (ab32576; Abcam), p-ERK (ab136926; Abcam), t-ERK (ab184699; Abcam), Vimentin (ab92547; Abcam), Bax (ab32503; Abcam), CyclinD1 (ab16663; Abcam) and GAPDH (ab181602; Abcam). The membranes were incubated at 4°C overnight, washed with TBST three times, five min each time. Then, the membranes were incubated with secondary antibodies for 1 h at room temperature. The membranes were washed with TBST three times, five min each time. Finally, ECL luminescent solution was added to the dark room, exposed and developed.
5
Exosome Isolation and Western Blot Analysis
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Exosomes were prepared using 1× RIPA buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% Triton-100, 1% sodium deoxycholate, BYLABS, Hanam, Republic of Korea). The exosome concentration was determined using bicinchoninic acid. We added 6X sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer to prepare the lysate solution. For sample preparation, equal amounts of protein samples were separated using 10% SDS-PAGE and transferred onto 0.45 µm nitrocellulose membranes (GE Healthcare Life Science, Port Washington, NY, USA). The membranes were incubated with 5% skim milk in Tris-buffered saline with 0.1% Tween® 20 Detergent, followed by immunoblotting with specific primary antibodies against CD9 (rabbit monoclonal, Abcam, Cambridge, UK, ab92726), CD81 (mouse monoclonal, sc-166029), glyceraldehyde-3-phosphate dehydrogenase (Santa Cruz, sc-137179, Dallas, TX, USA), E-cadherin (rabbit monoclonal, Abcam, ab92547), β-catenin (rabbit monoclonal, Abcam, ab32576), and N-cadherin (rabbit polyclonal, Abcam, ab18203), followed by appropriate secondary antibodies. Protein bands were visualized using an enhanced chemiluminescence (ECL) substrate (GE Healthcare Life Science) and analyzed using a LAS-3000 imager (GE Healthcare Life Science) and film.
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