(A) Immunoblot of ALK5 expressed in COS1 cells together with TβRII and stimulation or not with 2 ng/ml TGF-β1 for 12 h in the absence or presence of increasing wild-type GFP-SIK amounts

(B and C) Pulse-chase analysis of CA-ALK5, KR-ALK5, and CIN85 in the absence (−) or presence (+) of cotransfected SIK (S). A 30-min pulse was followed by chase for the indicated times. Treatment with proteasomal (LLnL 200 μM; S + L) or lysosomal (chloroquine 400 μM; S + Q) inhibitor during the chase is shown in C. Radioactive quantitation of ALK5 or CIN85 protein is plotted relative to the pulse signal. Representative data from three independent repeats are shown. Protein autoradiograms are shown below each graph. (D) Immunoblot of CA-ALK5 in COS1 cells cotransfected with wild-type and mutant SIK constructs. (E, top) quantitative real-time RT-PCR analysis of mRNA in HaCaT cells after transfection with control (siLuc) and specific (siSIK) siRNAs and 8 h of stimulation with 5 ng/ml TGFβ1. Mean fold induction of TGFβ-stimulated (black bars) relative to unstimulated levels (gray bars) are plotted, with standard errors determined from triplicate samples. (E, bottom) Immunoblot of endogenous SIK, ALK5, and α-tubulin loading control in HaCaT total cell lysates (TCL) prepared as per mRNA analysis. (F) Mv1Lu cells transfected with control (siLuc) or specific (siSIK) siRNA were incubated with radio-TGFβ1 before cross-linking and incubation at 37°C for the indicated time periods. Cell surface receptor autoradiograms show the three TGF-β receptors (TβR) marked as type I, II, and III, and the unbound ligand (TGFβ) migrating at the bottom. Protein sizes are in kilodaltons. Black lines indicate that intervening lanes have been spliced out.

Copyright information:

Taken from "TGFβ induces SIK to negatively regulate type I receptor kinase signaling"

The Journal of Cell Biology 2008;182(4):655-662.

Published online 25 Aug 2008

PMCID:PMC2518705.