Supplementary Material for DNAJB6 chaperones PP2A mediated dephosphorylation of GSK3-β to down regulate osteopontin expression. Aparna Mitra et al.

Cell culture: MDA-MB-435 cells were maintained as described before (Mitra et al.). MDA-MB-435 cells stably transfected with either full length DNAJB6 (Accession # NM_058246.3) or DNAJB6-ΔJ or DNAJB6-HPDMUT were grown in medum with 500 μg/ml G418 (Invitrogen, Carlsbad, CA, USA). Malignant human melanoma cell line, A375, was cultured in RPMI containing 10% FBS. COS7 cells were cultured in Dulbeco’s Modified Eagle Medium/F12. All cells were maintained at 37°C with 5% carbon dioxide in a humidified atmosphere. [Despite conflicting reports, MDA-MB-435 has now been confirmed to be of melanoma origin (Rae et al., 2007; Rae et al., 2004; Xie et al., 1992). However there still are arguments about the lineage infidelity of this cell line (Chambers, 2009)

Collection of serum free media and western blot for secreted proteins:

Cells (2×106) were plated in a 10 cm plate containing appropriate growth medium overnight. The growth medium was replaced with 3 ml serum-free phenol-free DMEM/F12. The conditioned medium was harvested 18 hours later, spun at 1,300 rpm at 4°C for 10 minutes. The media samples were resolved on SDS-PAGE and immunoblotted for proteins of interest. Serum free media was also given for Mass spectrometric analysis as a control.

Soft agar colonization assay:

Cells (2 × 103) suspended in 0.35% agar were plated onto a layer of 0.75% bactoagar in DMEM/F12 (5% fetal bovine serum) in six-well tissue culture dishes. Visible colonies (> 50 cells) were counted after 15 days with the aid of a dissecting microscope.

Immunoblotting:

Cells at 90% confluence lysed in NP-40 lysis buffer. Proteins (20 μg) were resolved by SDS-PAGE and immunoblotted for DNAJB6 (Abnova Corporation, Taipei City, Taiwan), OPN (Sigma, St. Louis, MO, USA or Assay Designs, Ann Arbor, Michigan,USA), β-actin (BioRad) or GSK3β, pGSK3β-ser9, PP2A, β-tubulin (Cell Signaling Technology, Danvers, MA, USA). Blots were developed using SuperSignal™ (Pierce, Rockford, IL, USA) and imaged on a Fuji LAS3000 imager or on Kodak film.

Immunoprecipitation:

Cells were lysed in NP-40 lysis buffer containing protease and phosphatase inhibitors (Calbiochem, Merck KGaA, Darmstadt, Germany). After incubation on ice for 1 hr, the lysate was centrifuged at 16,000xg for 15 min and 1 μg of appropriate antibody was added and incubated at 4°C on an orbital shaker for a minimum of 1 hr. Protein A/G PLUS Agarose beads (Santa Cruz Biotech) were added and samples incubated overnight at 4°C with mixing. The beads were washed 3 times in PBS + 0.01% NP-40. The proteins were eluted for SDS-PAGE by addition of denaturing and reducing sample buffer.