(a) HEK293T cells were transfected with the indicated constructs, lysed, and immunoblotted as indicated. Where indicated, cells were treated with MG132 for four hours prior to collection.
(b) HeLa cells were synchronized at G1/S by double-thymidine block before trypsinization and release into fresh media. Cells were transfected with either an siRNA targeting cyclin F or a non-targeting (N/T) siRNA between the first and second thymidine treatment, collected at the indicated time points, and immunoblotted as indicated.
(c) HEK293T cells were transfected with the indicated constructs. Whole cell extracts were immunoblotted as indicated. (l.e.: long exposure, s.e: short exposure).
(d) HEK293T cells were transfected with the indicated constructs. Whole cell extracts were immunoprecipitated with anti-FLAG resin and immunoblotted as indicated. The bracket indicates a ladder of bands corresponding to poly-ubiquitylated SLBP.
See also Figure S2