Whole cell extracts were prepared by lysing cells in Laemmli buffer containing 1% SDS and 2% 2-mercaptoethanol. Lysates from equal numbers of cells were separated by 8 or 10% SDS PAGE, transferred to PVDF membranes (GE Healthcare), incubated with primary antibodies and detected with HRP-conjugated species-specific anti-immunoglobulin light chain antibodies (Jackson ImmunoResearch) and a Luminata HRP substrate (Millipore). Anti-HDAC1 (Abcam, ab7028) and anti-Tubulin beta (Developmental Studies Hybridoma Bank, University of Iowa) were used as loading controls. Anti-AP4 antibody was previously described24 (link). The following antibodies were purchased: anti-c-Myc (Cell Signaling, 9402S), anti-phospho STAT5 (PY694) (BD Biosciences, 611964), anti-Blimp-1 (Genscript, A01647-40), anti-T-bet (Santa Cruz, sc-21003). For translation inhibition and proteasome inhibition, 10 μM of cycloheximide (Sigma) or MG-132 (Sigma) was added to the cell culture. For inhibition of signaling pathways, 20 nM of U0126 (Cayman Chemical), 10 μM of SB203580 (Cayman Chemical), 50 nM of wortmannin (Cayman Chemical), 5 nM of FK506 (Cayman Chemical), or 2.5 nM of rapamycin (Cayman Chemical) was added to the cell culture.
Anti blimp 1
Manufactured by GenScript
Anti-Blimp-1 is a lab equipment product manufactured by GenScript. It is designed to detect and measure the levels of Blimp-1 (B Lymphocyte-Induced Maturation Protein 1) in biological samples. Blimp-1 is a transcription factor that plays a crucial role in the differentiation of B cells into plasma cells. The Anti-Blimp-1 product allows researchers to investigate the expression and regulation of Blimp-1 in various biological systems.
Automatically generated - may contain errors
Lab products found in correlation
Rapamycin, by Cayman Chemical (2 mentions)
Wortmannin, by Cayman Chemical (2 mentions)
Anti c myc, by Cell Signaling Technology (2 mentions)
Anti phospho stat5 py694, by BD (2 mentions)
Pvdf membrane, by GE Healthcare (2 mentions)
Fk506, by Cayman Chemical (2 mentions)
Anti hdac1, by Abcam (2 mentions)
Luminata hrp substrate, by Merck Group (2 mentions)
Hrp conjugated species specific anti immunoglobulin light chain antibodies, by Jackson ImmunoResearch (2 mentions)
Anti beta tubulin, by Developmental Studies Hybridoma Bank (2 mentions)
U0126, by Cayman Chemical (2 mentions)
Sb203580, by Cayman Chemical (2 mentions)
Anti t bet, by Santa Cruz Biotechnology (2 mentions)
Mg132, by Merck Group (2 mentions)
Cycloheximide (chx), by Merck Group (2 mentions)
Ab7029, by Abcam (1 mentions)
Ab8898, by Abcam (1 mentions)
Ab6002, by Abcam (1 mentions)
Anti p300 sc 585, by Santa Cruz Biotechnology (1 mentions)
Anti cbp, by Cell Signaling Technology (1 mentions)
3 protocols using anti blimp 1
1
Western Blot Analysis of Protein Expression
2
Western Blot Analysis of Protein Expression
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Whole cell extracts were prepared by lysing cells in Laemmli buffer containing 1% SDS and 2% 2-mercaptoethanol. Lysates from equal numbers of cells were separated by 8 or 10% SDS PAGE, transferred to PVDF membranes (GE Healthcare), incubated with primary antibodies and detected with HRP-conjugated species-specific anti-immunoglobulin light chain antibodies (Jackson ImmunoResearch) and a Luminata HRP substrate (Millipore). Anti-HDAC1 (Abcam, ab7028) and anti-Tubulin beta (Developmental Studies Hybridoma Bank, University of Iowa) were used as loading controls. Anti-AP4 antibody was previously described24 (link). The following antibodies were purchased: anti-c-Myc (Cell Signaling, 9402S), anti-phospho STAT5 (PY694) (BD Biosciences, 611964), anti-Blimp-1 (Genscript, A01647-40), anti-T-bet (Santa Cruz, sc-21003). For translation inhibition and proteasome inhibition, 10 μM of cycloheximide (Sigma) or MG-132 (Sigma) was added to the cell culture. For inhibition of signaling pathways, 20 nM of U0126 (Cayman Chemical), 10 μM of SB203580 (Cayman Chemical), 50 nM of wortmannin (Cayman Chemical), 5 nM of FK506 (Cayman Chemical), or 2.5 nM of rapamycin (Cayman Chemical) was added to the cell culture.
3
Chromatin Immunoprecipitation of Blimp-1 and Histone Modifications
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CD4+ T cells were cross-linked for 10 minutes by adding formaldehyde to tissue culture medium; the final concentration of formaldehyde was 1%. Cross-linked cells were washed with phosphate-buffered saline containing protease inhibitors. Then the subsequent steps were performed using a commercial ChIP kit (MilliporeSigma). Finally, nuclear extraction was performed according to the manufacturer’s instructions. The lysates were sonicated for 20 cycles of 30 seconds each, resting on ice for 30 seconds between cycles. Cross-linked and sonicated chromatin was incubated overnight with anti–Blimp-1 (A01647, GenScript), anti–acetylated H3 (06-599, MilliporeSigma), anti–acetylated H3 (K9) (07-352, MilliporeSigma), anti–trimethyl-histone H3 (K4) (17-641, MilliporeSigma), anti–acetylated H4 (06-866, MilliporeSigma), anti-CBP (7389, Cell Signaling Technology), anti-p300 (sc-585, Santa Cruz Biotechnology), anti–trimethyl-histone H3 (K9) (ab-8898, Abcam), anti–trimethyl-histone H3 (K27) (ab-6002, Abcam), anti-HDAC2 (ab-7029, Abcam), anti-V5 (13-202, Cell Signaling Technologies), and anti-HA (A190-108A, Bethyl Laboratories) antibodies. DNA fragments were recovered using a DNA purification kit provided in the ChIP kit (MilliporeSigma) and analyzed by quantitative PCR using specific primers (Supplemental Table 2 ). Samples from at least 3 independent immunoprecipitations were analyzed.
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