Amersham imager 600 digital imagingsystem

Ten micrograms of protein from each sample
was mixed with 5× Laemmli buffer with 5% β-mercaptoethanol,
heated for 5 min at 100 °C, and separated on 10% sodium dodecyl
sulfate (SDS) polyacrylamide gel electrophoresis gels. Following electrophoresis,
the proteins were transferred to Immobilon-P transfer membranes (Sigma-Aldrich)
using the Trans-Blot TurboTM Transfer System (Bio-Rad Laboratories).
Membranes were visualized with Ponceau Red (FlukaTM Analytical). Membranes
were probed with the following antibodies purchased from Cell Signaling
Technology: MAPKAPK3 (#7421), MAPK14 (#9218), Thr180/Tyr182-P-p38
MAPK (#4511), and anti-rabbit IgG-HRP (#7074). GAPDH (sc-47,724) was
purchased from Santa Cruz. The Amersham Imager 600 digital imaging
system (GE Healthcare) was used for image acquisition and Quantity
One v4.6.3 (Bio-Rad Laboratories) for band densitometry. All western
blots are presented as cropped images, with full scans of blots provided
in Figure S4.

Cells were lysed using 5% SDS supplemented
with the Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail
2 (Sigma-Aldrich). The following antibodies were purchased from Cell
Signaling Technology (Danvers): MK2 (#3042), Thr-334-P-MK2 (#3041),
MK3 (#7421), MKK3 (#8535), Ser189-P-MKK3/Ser207-P-MKK6 (#12280), p38
MAPK (#9212), p38α MAPK (#9218), p38γ MAPK (#2307), Thr180/Tyr182-P-p38
MAPK (#4511), MPP9 (#13667), anti-rabbit IgG-HRP (#7074), and anti-mouse
IgG (#7076). GAPDH (sc-47724) was from Santa Cruz, and α-tubulin
(T9026) was from Sigma-Aldrich. The Amersham Imager 600 digital imaging
system (GE Healthcare) was used for image acquisition.