1 × 106 cells/mL (5 × 106 cells/mL for CLL primary cells or healthy PBMCs) were seeded in 2 mL per well of a 6-well plate (Corning) and exposed to a range of concentrations of E7107 and/or idasanutlin. Concentrations are indicated in the figure legends. Protein lysates were harvested using 2% SDS lysis buffer at 24 h, heated at 95 °C for 10 min, and sonicated. Protein concentration was measured using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, UK). Primary antibodies against p53 (DO-7) (#M7001, Dako), MDM2 (Ab-1) (#OP46, Merck Millipore), p21WAF1 (Calbiochem), PARP (Trevigen), SF3B1 (Sigma), Actin (Sigma), Phospho-Rb (Ser780) (Cell Signalling), Phospho-Rb (Ser807/811) (Cell Signalling), Phospho-Rb (T821) (Abcam) and secondary goat anti-mouse/rabbit horseradish peroxidase-conjugated antibodies (Dako) were used. All antibodies were diluted in 5% (w/v) nonfat milk or BSA in TBS-tween20. Proteins were visualized using enhanced chemiluminescence reagents (GE Healthcare).
Sf3b1
Manufactured by Merck Group
SF3B1 is a component of the spliceosome, a large molecular complex that is responsible for the splicing of pre-mRNA to mature mRNA. It plays a crucial role in the splicing process by regulating the recognition of the 3' splice site during the formation of the spliceosome.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Enhanced chemiluminescence reagent, by GE Healthcare (2 mentions)
Secondary goat anti mouse rabbit horseradish peroxidase conjugated antibodies, by Agilent Technologies (2 mentions)
Phospho rb ser807 811, by Cell Signaling Technology (1 mentions)
Phospho rb ser780, by Cell Signaling Technology (1 mentions)
6 well plate, by Corning (1 mentions)
Superscript 3 first strand synthesis supermix, by Thermo Fisher Scientific (1 mentions)
Srsf1, by Merck Group (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Rna isolation kit, by Qiagen (1 mentions)
Itaq universal sybr green supermix, by Bio-Rad (1 mentions)
Cfx connect real time pcr detection system, by Bio-Rad (1 mentions)
Mdm2 ab 1, by Merck Group (1 mentions)
3 protocols using sf3b1
1
Protein Expression Analysis in Cells
2
Knockdown of Spliceosome Proteins
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siRNAs against human SANS (IDT), PRPF6 (Sigma), PRPF31 (IDT), SRSF1 (Sigma) SF3B1 (Sigma) and SON (Dharmacon) were purchased as indicated in Supplementary Table S3 . Non-targeting control siRNA (siCtrl) was purchased from IDT. For knockdowns, HEK293T and HeLa cells were transfected with a final concentration of 20 nM siRNAs using Lipofectamine RNAiMAX (Invitrogen) following the manufacturer's protocol. Subsequently, total RNA was isolated from transfected cells using a Qiagen RNA isolation kit and converted to cDNA with SuperScript™ III First-Strand Synthesis SuperMix (ThermoFisher) according to manufacturer's protocols by using Oligo (dT) primers. For the validation of knockdowns, quantitative PCR (qPCR) was performed using gene specific primers (Supplementary Table S6 ) and iTaq Universal SYBR Green Supermix (Bio-Rad). Cq values were detected with a CFX Connect Real-Time PCR Detection System (Bio-Rad). Gene expression profiles were calculated with ΔΔCq methods based on normalization to GAPDH (for primers, see Supplementary Table S6 ). SANS siRNA knockdown efficiency was also validated by Western blotting and immunofluorescence analysis in HEK293T and HeLa cells (Supplementary Figure S3 ).
3
Western Blot Analysis of p53 Pathway
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The 1 × 106 cells/mL (5 × 106 cells/mL for primary CLL cells) were seeded in 2 mL per well of a 6-well plate (Corning) and subjected to the corresponding manipulation (siRNA transfection and/or exposure to siremadlin or idasanutlin). Protein lysates were harvested using 2% SDS lysis buffer at 24 h, heated at 95 °C for 10 min, and sonicated. Protein concentration was measured using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Primary antibodies against p53 (DO-7) (#M7001, Dako), p21WAF1 (Calbiochem), SF3B1 (Sigma), MDM2 (Ab-1) (#OP46, Merck Millipore), Actin (Sigma) and secondary goat anti-mouse/rabbit horseradish peroxidase-conjugated antibodies (Dako) were used. All antibodies were diluted in 5% (w/v) non-fat milk or BSA in TBS-tween20. Proteins were visualized using enhanced chemiluminescence reagents (GE Healthcare, Chicago, IL, USA).
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