Mdia1

All cell lines were obtained from American Type Culture Collection. MCF10A cells were cultured as described (Debnath et al., 2003). MCF7 and HEK293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) plus 10% heat inactivated FBS. Cell Dissociation Buffer, enzyme-free, PBS-based (Life Technologies). Oligonucleotides of small interfering RNA (siRNA) for LPA receptors, G protein alpha subunits, and RhoGEFs were synthesized by QIAGEN. Oligonucleotides of siRNA for mDia1 were purchased from IBA GmbH. LPA (1-Oleoyl Lysophosphatidic Acid) and EDG family inhibitor Ki16425 were purchased from Cayman Chemical Company. Poly (2-hydroxyethyl methacrylate) (PolyHEMA) was purchased from Polysciences Inc. Antibodies against EDG4 were from Assay Biotechnology; LPAR receptors, Ezrin and LARG from Santa Cruz Biotechnology; PDZ-RhoGEF from IMGENEX; pMLC2 from Sigma and mDia1 from BD Biosciences. pCMV6-XL5 LPAR2 expression vector were purchased from OriGene (SC117226). pWPXL-based lentiviral expression vectors for H2B, LPAR2, Gα12, and Gα12Q/L were generated using standard PCR-based procedures or in the case of LifeAct-GFP were a kind gift from Oliver Fackler. Delipidized FCS was from Bio&SELL e.K.

A total of 15–50 oocytes were lysed in RIPA buffer (GenDOPET, Texas, USA) containing protease inhibitors and heated for 5 min at 100 °C. Total oocyte proteins were subjected to electrophoresis on a 10% SDS-PAGE gel. The separated proteins were transferred to PVDF membranes, which were pretreated with methanol. The membranes were blocked in 5% skim milk and incubated with primary antibodies as follows: HDAC6 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), Acetyl-α-tubulin (Thermo Fisher Scientifc, Rockford, IL, USA), mDia1 (BD Biosciences, San Jose, CA), and mTOR (2983), PI3 Kinase p110α (4249), Phospho-Akt (4060), Akt (4685), Phospho-p44/42 (9101) and p44/42 (9102), all purchased from Cell Signaling Technology (Beverly, MA, USA). After three washes in TBST, the blots were then incubated with anti-rabbit or anti-mouse IgG antibody conjugated to horseradish peroxidase for 1 h. The protein bands were visualised using an SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientifc, Rockford, IL, USA). The membrane was then washed and reblotted with an actin antibody as an internal control. Densitometric quantification was performed using the ImageJ software (NIH, Bethesda, Maryland).