For sample preparation, when the cell culture reaches to 80% confluence, the cells were collected and split for 40 minutes on ice, the supernatant was denatured in 5x loading buffer containing SDS in boiling water for 10 minutes. 5% non-fat dry milk dissolved in TBST (TBS containing 0.1% Tween-20) was used for blocking and antibody dilution. The following antibodies and dilutions were used: ZEB1 (1:500, 21544-1-AP, Proteintech), VIM (1:1000, 5741s, Cell Signaling Technology), E-CAD (1:1000, 20874-1-AP, Proteintech), actin (1:1000, HC201, TransGen), MSN (1:2000, EP1863Y, abcam), goat anti-mouse IgG-HRP (1:5000, sc-2005, Santa Cruz), goat anti-rabbit IgG (1:5000, sc-2004, Santa Cruz). Western HRP Substrate (WBLUF0500, Millipore) was used to detect horseradish peroxidase conjugated secondary antibodies.
Ep1863y
Manufactured by Abcam
EP1863Y is a monoclonal antibody that recognizes the BRCA1 protein. It is intended for use in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti mouse igg hrp, by Santa Cruz Biotechnology (1 mentions)
Goat anti rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Hc201, by Transgene (1 mentions)
Macs protein a microbeads, by Miltenyi Biotec (1 mentions)
Streptavidin microbeads, by Miltenyi Biotec (1 mentions)
Soluble anti cd28 37.51, by Cytek Biosciences (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Pan t cell isolation kit 2, by Miltenyi Biotec (1 mentions)
96 well plate, by Sarstedt (1 mentions)
Zli 9610, by ZSGB-BIO (1 mentions)
4 protocols using ep1863y
1
Cell Culture Preparation for Western Blot
2
Moesin and gpALL Pulldown Assays
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For moesin immunoprecipitation experiments, 5 µl of unconjugated anti-moesin mAb (EP1863Y, Abcam) was mixed with 200 µl of the T cell protein extract (200 µg/ml) and incubated overnight at 4°C; then, 50 µl of µMACS Protein A Microbeads (Miltenyi Biotec) were added (2 h, 4°C). The immunoprecipitate’s washing, elution, and separation were performed according to the manufacturer’s recommendations. For gpALL pull-down, 200 µl of the T cell protein extract (200 µg/ml) was mixed with 30 µL of biotinylated ALL (2.2 mg/ml) and incubated at 4°C overnight; then, 100 µl of Streptavidin MicroBeads (Miltenyi Biotec) were added and further incubated for 2 h, at 4°C; the precipitate’s elution, washing, and separation were performed with 500 mM GalNAc following manufacturer’s instructions. The precipitates were used in later experiments.
3
T Cell Proliferation Assay with Stimulants
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Splenocytes were obtained by perfusion with DPBS and red cells were removed using hypotonic NH4Cl lysing buffer. Cells were stained with CFSE (Thermo Fisher) as previously reported (36 (link)) and T lymphocytes were purified by negative selection using the Pan T cell Isolation Kit II (Miltenyi Biotec). Cells (2.5 × 105 cells/ml) were incubated in complete RPMI medium (RPMI 1640 supplemented with 2 mM L-glutamine, 10 mM non-essential amino acids, 1 mM sodium pyruvate, 25 mM HEPES, 50 mM 2-β mercaptoethanol, and 50 U/ml penicillin-streptomycin from GIBCO and 10% FCS (ByProducts) in 96-well plates (Sarstedt) at 37°C, 5% CO2. Cells were stimulated with 3 µg/ml soluble anti-CD3 (145-2C11, in house purified) and 3 µg/ml soluble anti-CD28 (37.51; Tonbo Biosciences), 5 µg/ml ALL, and/or 5 µg/ml anti-moesin (EP1863Y, Abcam). Seventy-two hours later cells were harvested, washed (DPBS, 1% FCS), and stained with the corresponding mAb combination.
4
Immunohistochemical Analysis of Tissue Samples
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The tumor tissues or the livers of mice were fixed in formalin and processed for paraffin embedding. Sectioned samples were de-paraffined in xylene and rehydrated in graded alcohol. Antigen retrieval was done according to the manufacture's protocol (MVS-0100, Maxvision), and then the endogenous peroxidase was inactivated with 3% hydrogen peroxide methanol solution, blocked with animal non-immune serum (SP KIT-B, Maxvision) and incubated with primary antibodies overnight at 4℃, and then incubated with secondary antibodies for 15 minutes. Slides were stained using the detection kit (DAB-0031, Maxvision), cell nucleus was stained with hematoxylin (ZLI-9610, ZSGB-BIO). The following antibodies and dilutions were used for immunohistochemistry: MSN (1:150, EP1863Y, Abcam), peroxidase-conjugated secondary antibody (KIT-5010, Maxvision), GFAP (ZM-0118, ZSGB-BIO), AFP (ZM-0009, ZSGB-BIO).
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