aHSCs were treated as indicated, detached, thoroughly washed with PBS, and then lysed in ice-cold lysis buffer. Following centrifugation at 13,000 ×g for 10 min at 4°C, the supernatants (30 μg protein) were boiled with reducing sample buffer for 5 min, subjected to electrophoresis in SDS-polyacrylamide gels, and then transferred onto a PVDF membrane. The membrane was blocked with 1% BSA in PBS containing 0.1% Tween-20 (PBST) for 1 h at room temperature and then washed with PBST. Proteins were detected by incubating the membrane overnight at 4°C with antibodies against α-SMA, β-actin (Sigma-Aldrich, MO), Bak, Bal-2 (C21), Bax, caspase-3 (H-277), caspase-9 (H-170) (Santa Cruz Biotechnology, CA), cleaved caspase-9 (Asp 353), and cleaved PARP (Asp 214) (Cell Signaling Technology, MA). Next, the membrane was incubated with a primary antibody, and, finally, the membrane was incubated with a secondary antibody conjugated to horseradish peroxidase (HRP) for 1 h. An enhanced chemiluminescence (ECL) kit (Amersham Biosciences, IL, or Millipore, MA) was used for protein detection. The relative intensity of the immunoreactive bands was assessed using Image J software.
Caspase 3 h 277
Manufactured by Santa Cruz Biotechnology
Sourced in Macao
Caspase-3 (H-277) is a primary antibody that recognizes the Caspase-3 protein. Caspase-3 is a key enzyme involved in the execution phase of cell apoptosis (programmed cell death).
Automatically generated - may contain errors
Lab products found in correlation
Cleaved caspase 9 asp353, by Cell Signaling Technology (1 mentions)
Ecl kit, by Cytiva (1 mentions)
Cleaved parp asp214, by Cell Signaling Technology (1 mentions)
Caspase 9 h 170, by Santa Cruz Biotechnology (1 mentions)
α sma, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
Control sirna a, by Santa Cruz Biotechnology (1 mentions)
Myc 9e10, by Santa Cruz Biotechnology (1 mentions)
Myc y69, by Abcam (1 mentions)
Snai1 sirna, by Santa Cruz Biotechnology (1 mentions)
Control sirna, by Horizon Discovery (1 mentions)
Puromycin, by Avantor (1 mentions)
Aprotinin, by Avantor (1 mentions)
Leupeptin, by Avantor (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Avantor (1 mentions)
Gapdh, by US Biological (1 mentions)
Stat6 d3h4, by Cell Signaling Technology (1 mentions)
Hygromycin b, by Merck Group (1 mentions)
Parp1 f 2, by Santa Cruz Biotechnology (1 mentions)
P stat6 tyr641, by Cell Signaling Technology (1 mentions)
3 protocols using caspase 3 h 277
1
Western Blot Analysis of Apoptosis Markers
2
MYC Acetylation Regulation Assay
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The antibodies used were MYC Y69 from Abcam; MYC N-262, MYC C-33, MYC 9E10, MAX C-17, p300 N-15, GCN5 N-18, TRRAP T-17, and caspase-3 H-277 from Santa Cruz Biotechnology; cleaved caspase-3 and acetylated lysine from Cell Signaling Technology; vinculin, FLAG/M2, and FLAG M2 affinity resin from Sigma; TBP, which was a gift from Dr. Robert. G. Roeder; and TIP49, which was a gift from Dr. Bruno Amati. The affinity-purified site-specific MYC AcK antibodies were generated in rabbits by immunization with specific acetylated peptides (Supplemental Fig. S1E ) in collaboration with EMD Millipore and are commercially available as acetyl-c-MYC (K148) ABE25, acetyl-c-MYC (K157) ABE27, and acetyl-c-MYC (K323) ABE26 (EMD Millipore). The plasmid DNAs are described in the Supplemental Material . For RNAi, HeLa cells were transfected in 10-cm plates with Lipofectamine 2000 and 7.5 μg of either pCbS-FLAG-mMYC WT or the indicated R mutants and with 400 pmol of either a specific p300 siRNA or a control siRNA (Dharmacon). MCF10A-MYC cells in six-well plates were transfected with Lipofectamine 3000 and either 60 pmol of SNAI1 siRNA (Santa Cruz Biotechnology sc-38398) or control siRNA-A (Santa Cruz Biotechnology sc-37007). For further details, see the Supplemental Material .
3
Comprehensive Antibody Validation Protocol
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Antibodies to STAT6 (D3H4, Cell Signaling technology), p-STAT6 (Tyr-641, Cell Signaling Technology), FLAG (M2, Sigma), p53 (Do-1, Santa cruz), PARP1 (F2, Santa cruz), Caspase-3 (H-277, Santa cruz), TRIML2 (ARP55636-P050, Aviva System Biology), EBV ZEBRA (sc-53904, Santa cruz), ICP0 (11060, Santa cruz), Ub-K48 (05–1307, EMD Millipore),Ub-K63 (05–1308, EMD Millipore), LC3B (GT3612,GeneTex) and GAPDH (G8140-01, US Biological) were used according to the manufacturers specifications. The monoclonal antibody anti-myc (9E10) and HA (12CA5) were prepared from hybridoma cultures. Mouse monoclonal antibodies against RTA were kindly provided by Ke Lan from Shanghai Pasteur Institute of CAS. Antibodies against IE1/2 were kindly provided by Zhikang Qian from Shanghai Pasteur Institute of CAS. Tetradecanoyl Phorbol Acetate (TPA) was purchased from Sigma and sodium butyrate from J&K Corporation. Proteasome inhibitor MG132 was purchased from Biomol International. 3-Methyladenine (3-MA), Choloquine (Chl), Cyclohexamide (CHX) and Hygromycin B were purchased from Sigma–Aldrich. Doxycycline (DOX) was purchased from Sangon Biotech (Shanghai). PMSF, Leupeptin, Aprotinin, Pepstatin A and Puromycin were purchased from Amresco, and G418 from Inalco S.p.A.
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