siRNA transfections were performed on 3 × 105 to 4 × 105 cells in 35-mm culture dishes with antibiotic-free McCoy's 5A medium supplemented with 10% FBS and DharmaFECT2 transfection reagent (Dharmacon, Inc., no. T-2002-01). On-Target Plus siMcl-1, siBcl-xL, or siControl (Dharmacon, Inc., nos. L-004501-00, L-003458-00, and D-001810-10-05) was suspended in 1× siRNA buffer (Dharmacon, Inc., no. B-002000-UB-100) and added to a plate at a final concentration of 22 nM. Cells transfected with either siBcl-xL or siMcl-1 individually were harvested 48–72 h after transfection for Western blot analysis. Double siRNA knockdown (Bcl-xL and Mcl-1) was performed sequentially. Cells were first transfected with siBcl-xL or siControl. Forty-eight hours later, cells were split 1:2, with one half transfected with Mcl-1 siRNA and the other with siControl. Twenty-four hours later, cells from the double-knockdown plates and the siControl plates were harvested for Western blot analysis with PARP antibody or stained by Hoechst dye for quantification of the condensed nuclei.
Sibcl xl
Manufactured by Horizon Discovery
Sourced in United States
SiBcl-xl is a lab equipment product designed for specific research applications. It serves as a tool for researchers to conduct their experiments, but a detailed description of its core function cannot be provided while maintaining an unbiased and factual approach. Further information may be available from the product manufacturer or technical experts.
Automatically generated - may contain errors
Lab products found in correlation
Dharmafect 2 transfection reagent, by Horizon Discovery (2 mentions)
On target plus simcl 1, by Horizon Discovery (2 mentions)
Sicontrol, by Horizon Discovery (1 mentions)
Sirna buffer, by Horizon Discovery (1 mentions)
Amaxa cell line nucleofector kit l, by Lonza (1 mentions)
On targetplus, by Horizon Discovery (1 mentions)
Sirna duplexes, by RiboBio (1 mentions)
Sidesign, by Horizon Discovery (1 mentions)
5 protocols using sibcl xl
1
Simultaneous Knockdown of Bcl-xL and Mcl-1
2
Bcl-xl siRNA Synthesis and Preparation
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Non-fluorescent and fluorescein (FITC)-labeled anti-Bcl-xl siRNAs (siBcl-xl) (sense: 5′CAGGGACAGCAUAUCAGAGdTdT3′, antisense: 5′CUCUGAUAUGCUGUCCCUGdTdT3′) were synthesized (Dharmacon Inc., Lafayette, CO, USA) and dissolved in RNase-free water prior to experimental use.
3
Dual Knockdown of Bcl-xL and Mcl-1 in Cells
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Cells in 35-mm culture dishes with antibiotic-free McCoy's 5A medium+10% fetal bovine serum and DharmaFECT2 transfection reagent (Dharmacon, Inc., Lafayette, CO, USA, #T-2002-01) were subjected to siRNA transfection. siRNA Buffer (Dharmacon, Inc., #B-002000-UB-100) was used to suspend ON-TARGET Plus siMcl-1, siBcl-xl, or siControl (Dharmacon, Inc., #L-004501-00, #L-003458-00, #D-001810-10-05) at a final concentration of 22 nM. Transfected cells were harvested 48–72 h after transfection for western blot analysis. Sequential transfections were used for double knockdown of Bcl-xl and Mcl-1. The first transfection in cells was with siBcl-xl or siControl. Cells were split into two separate plates 48 h later. For the second transfection, one plate was transfected with siControl RNA and the other with siMcl-1 RNA. Cells subject to double knockdown and the siControl were harvested for western blot analysis with PARP antibody 24 h after the second transfection.
4
Bcl-XL Knockdown in Riva VR Cells
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Riva VR cells were transfected using the Amaxa® Cell Line Nucleofector® Kit L (Lonza, Basel, Switzerland), program C-05, as described in Bittremieux et al. [29 (link)]. Briefly, 3 × 106 cells were transfected with 500 nM siCTRL (ON-TARGET plus, non-targeting control pool, from Dharmacon) and 500 nM siBcl-XL (hs.Ri.BCL2L1.13.1, from IDT). At 24 h post-transfection, the cells were used for experiments and collected for Western blot analysis to confirm knockdown of Bcl-XL.
5
Optimized siRNA Constructs for Cellular Signaling
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All small interfering RNA (siRNA) duplexes were purchased from RIBOBIO (Guangzhou, China). Small interference RNAs (siRNAs) targeting human ATF4 (NM_001675), ATF6 (NM_007348), BAX (NM_001291428), BAK (NM_001188), BCL-xL (NM_138578), CHOP (NM_001195057), IRE1α (NM_001433), GOLGA2P10 (NR_026811), and PERK (NM_004836) are designed using the online tool siDESIGN (Dharmacon, IL, USA) and designated as siATF4, siATF6, siBAX, siBAK, siBCL-xL, siCHOP, siIRE1α, siP10, and siPERK, respectively. The negative control RNA duplex (NC) for siRNAs is non-homologous to any human genome sequence.
The firefly luciferase reporter vectors P(−1222/+175), P(−435/+175), P(−201/+175), P(−125/+175), and P(∆C) for GOLGA2P10 promoter activity assays, the ORF–GFP fusion protein expression vectors ORF–GFP, GAPDH–GFP and del-ATG–GFP for evaluating the coding ability of GOLGA2P10, the wild type or Ser75Ala mutant BAD expression constructs, and the lentiviral vectors for expressing human GOLGA2P10 and BCL-xL were constructed as described in Supplementary Materials and Methods. The sequences of siRNAs and primers for vector construction are listed in Supplementary Table2 .
The firefly luciferase reporter vectors P(−1222/+175), P(−435/+175), P(−201/+175), P(−125/+175), and P(∆C) for GOLGA2P10 promoter activity assays, the ORF–GFP fusion protein expression vectors ORF–GFP, GAPDH–GFP and del-ATG–GFP for evaluating the coding ability of GOLGA2P10, the wild type or Ser75Ala mutant BAD expression constructs, and the lentiviral vectors for expressing human GOLGA2P10 and BCL-xL were constructed as described in Supplementary Materials and Methods. The sequences of siRNAs and primers for vector construction are listed in Supplementary Table
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