Pools of small interfering RNA (siRNA) oligonucleotides directed against Rad54 (sc-36363), DNA-PKcs (sc-35201), or STAT3 (sc-29493) and control RNA (sc-37007) were purchased from Santa Cruz Biotech Inc. (Santa Cruz, CA, USA). Human (Hsa)-miR-21–5p (same sequence as mouse miR-21–5p) inhibitor was purchased from Ambion Inc. (Austin, TX). WT MEFs (control for Rad54 deficient cells (shown in Fig. 2a ) and an additional WT line obtained from Dr Gloria Li’s lab (shown in Supplementary Fig. 2 )) were transfected with oligonucleotides using Lipofectamine 3000 (Invitrogen, San Diego, CA, USA) according to the manufacturer’s instructions. Cells were then collected at 24–48 h after transfection and whole cell lysates were prepared in RIPA buffer as described previously [3 (link)]. Protein levels were determined using antibodies (against Rad54 (D-18), DNA-PKcs (H-163), EGFR (sc-03), STAT3 (sc-482) or actin (sc-47778)) that were purchased from Santa Cruz Biotech Inc. Antibodies against AP-1 (c-Jun) (#9165), phosphorylated EGFR (#3777) and ATM (#2873) were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). A phosphorylated ATR antibody (ABE462) was purchased from Millipore-Sigma Inc. (Billerica, MA, USA). The ATM inhibitor (KU-55933 (S1092)), ATR inhibitor (VE821 (S8007)), and EGFR inhibitor (gefitinib (ZD1839)) were purchased from Selleckchem (Houston, TX, USA).
Sc 29493
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-29493 is a laboratory equipment product offered by Santa Cruz Biotechnology. It is designed for scientific research purposes. The core function of this product is to provide a tool for researchers to conduct their experiments and studies.
Automatically generated - may contain errors
Lab products found in correlation
Gefitinib zd1839, by Selleck Chemicals (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Atm 2873, by Cell Signaling Technology (1 mentions)
Sirna oligonucleotides, by Santa Cruz Biotechnology (1 mentions)
Sc 482, by Santa Cruz Biotechnology (1 mentions)
Sn 1002, by Bioneer (1 mentions)
Neon transfection system, by Thermo Fisher Scientific (1 mentions)
Pulse controller, by Bio-Rad (1 mentions)
Sc 37007, by Santa Cruz Biotechnology (1 mentions)
Recombinant human il 11, by R&D Systems (1 mentions)
Scrambled sirna, by Santa Cruz Biotechnology (1 mentions)
Jetprime, by Polyplus Transfection (1 mentions)
5 protocols using sc 29493
1
Knockdown of DNA Repair Proteins
2
Inhibition of STAT3 in U266 Cells
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To inhibit STAT3 expression in U266 cells by RNA interference, U266 cells (2 × 106 cells/well) were transfected with 50 nM STAT3 siRNA (sc-29493; Santa Cruz Biotechnology) or 100 nM scrambled siRNA (SN-1002; BIONEER, Daejeon, Korea) using NEON Transfection system (Invitrogen). Then cells were incubated in 10% FBS-supplemented RPMI 1640 medium for 48 h.
3
STAT3 and p53 Knockdown Assay
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1.5 × 106 U373 cells were transfected with specific STAT3 siRNA transfection (Santa Cruz Biotechnology, sc-29493) and control siRNA-A (Santa Cruz Biotechnology, sc-37007) as a scrambled control for STAT3 knockdown, or with sip53 plasmid and empty vector as control for p53 knockdown, by electroporation using the Bio-Rad Pulse Controller at 180 V, according to the manufacturer's instructions, and cultured in 24 well plates. After 48 h cells were lysate and protein extracts were subjected to western blot analysis.
4
Targeting IL-11 and STAT3 in Research
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Small interfering RNAs against IL-11 (sc-39636) or STAT3 (sc-29493) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Recombinant human IL-11 and IL-11 neutralizing antibody were purchased from R&D Systems (Minneapolis, MN, USA). Recombinant human IL-11 was reconstituted in PBS containing 0.5% bovine serum albumin (BSA) before being used in experiments.
5
MDSC Transfection with STAT3 siRNA
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Isolated MDSCs were transfected with siRNA using transfection reagent JetPRIME (Polyplus, Illkirch, France), at the final concentration (60 pmol) according to the manufacturer’s recommendations. For siRNA transfection of MDSCs, cells were plated onto a 24-well plate and incubated at 37°C overnight. Then, siRNAs and the reagents of siRNA transfection were subsequently diluted in siRNA transfection medium (sc-29493, Santa Cruz Biotechnology, CA, USA) individually. Afterward, they were incubated at room temperature (25°C) for 5 min. The supplied solutions were then mixed and incubated at room temperature (25°C) for 30 min. Before transfection, the medium was changed to Opti-MEM Medium, and then the mixtures were added to the cells in a drop-wise manner. Cells were incubated at 37°C for 5–6 h in a humidified atmosphere of 5% CO2, and then RPMI-1640 medium containing 20% FBS was added. The used sample of STAT3 siRNAs in this section of work included three different siRNA duplexes. Scrambled siRNA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was considered as a negative control for the experiments (Supplementary Table S1
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