Article Title: Regulation of VCP/p97 demonstrates the critical balance between cell death and epithelial-mesenchymal transition (EMT) downstream of ER stress
Figure Lengend Snippet: Loss of VCP induces ER stress A. Western blot analysis of different proteins involved in unfolded protein response and ER stress response. Cells were transfected with either non targeting (siNT) siRNA or two different siRNAs targeting VCP (siVCP1 and siVCP2). After 72 hrs of transfection, cells were harvested and analyzed for protein expression. B. Fluorescence staining for Chop and Calnexin in A549 and H358 cells. After 24 hrs of transfection either with non-targeting siRNA (siNT) or with siRNAs targeting VCP (siVCP1 or siVCP2) cells were trypsinized and plated on chamber slides. After 48 hours cells were fixed and stained for Chop and Calnexin. i, iii and v: Chop was detected using Alexa Fluor 488 rabbit anti-mouse IgG (green). ii, iv and vi: overlay of respective Chop and F-actin (Alexa Fluor 568 Phalloidin; red) staining with DAPI counter stain. a, c and e: Calnexin was detected using Alexa Fluor 488 goat anti-rabbit IgG (green). b, d and f: overlay of respective Calnexin and F-actin (Alexa Fluor 568 Phalloidin; red) staining with DAPI counter stain.
Article Snippet: Antibodies used for study VCP #2649, Bip #3183, CHOP #2895, eIF2α #9722 and p-eIF2α #9721, IRE1α #3294, PERK #5683, JNK #9258, p-JNK 4668, p38 #8690, P-p38 #4511, LC3 #3868, Cleaved Caspase3 #9664, Calnexin #2679, PDI #3501, E-cadherin #3195, Vimentin #5741, Zeb1 #3396, Claudin1 #4933, Snail #3879, Slug #9585, Integrin β3 #4702, Akt #9272, P-Akt #9271, mTOR #2972, P-mTOR #2971 (Cell Signaling Technologies Inc. Danvers, MA 01923); GAPDH #FL335 (Santa Cruz); Alexa Fluor 488 goat anti-rabbit IgG #A11034 and Alexa Fluor 546 goat anti-rabbit IgG #A11010 (Molecular Probes, Invitrogen detection technologies, Eugene, OR.
Techniques: Western Blot, Transfection, Expressing, Fluorescence, Staining