After treatment of S2 cells with either clamp or gfp RNAi, we collected a total of 2mL of cells for total RNA (1mL cells) and protein extraction (1mL cells). The preparation of mRNA for qPCR analysis was performed as previously described [7 (link),11 ], with the exception that gapdh was used for internal normalization. Primers used to target amplification have been published previously [7 (link),31 (link)]. The average ΔCt values for clamp or nelf-b transcript was calculated from four biological replicates and significant differences between means were calculated using a T-test.
Total protein was extracted to determine NELF-B abundance after clamp RNAi following the protocol described previously [7 (link),11 ]. Immobilized proteins were blotted for NELF-B (rabbit, 1:1000, gift from Karen Adelman) and detected using the Western Breeze kit (ThermoFisher Scientific). A similar protocol was followed to detect associations between CLAMP and NELF-B after immunoprecipitation. For the detection of NELF-A, proteins were transferred to PVDF membrane using the Xcell IITM blot module. The Western Breeze kit was then used to detect NELF-A (rabbit, 1:1000, gift from David Gilmour).
Total protein was extracted to determine NELF-B abundance after clamp RNAi following the protocol described previously [7 (link),11 ]. Immobilized proteins were blotted for NELF-B (rabbit, 1:1000, gift from Karen Adelman) and detected using the Western Breeze kit (ThermoFisher Scientific). A similar protocol was followed to detect associations between CLAMP and NELF-B after immunoprecipitation. For the detection of NELF-A, proteins were transferred to PVDF membrane using the Xcell IITM blot module. The Western Breeze kit was then used to detect NELF-A (rabbit, 1:1000, gift from David Gilmour).
Free full text: Click here
