Journal: The Journal of Cell Biology
Article Title: Rab5c promotes AMAP1-PRKD2 complex formation to enhance ?1 integrin recycling in EGF-induced cancer invasion
Figure Lengend Snippet: Association of PRKD2 with AMAP1 in highly invasive breast cancer cells. (A) Lysates from different breast cancer cells and HMECs were probed with the indicated antibodies. (B) Lysates from the indicated cells were immunoprecipitated with an anti-AMAP1 antibody or an anti-AMAP2 antibody, then subjected to blotting with the indicated antibodies. Preimmune serum of the anti-AMAP1 antibody was used as a control (Pre-imm). WCL, whole cell lysate (5 µg). (C and D) MDA-MB-231 cells, expressing mVenus-PKD2 and cultured on Alexa Fluor–labeled gelatin film, were fixed and stained with an anti-AMAP1 antibody. Original colors were: PRKD2, green; AMAP1, red; and sites of gelatin degradation, blue (C). Bar, 10 µm. Red intensities (AMAP1, arbitrary units) and green intensities (PRKD2, arbitrary units) at the sites of gelatin degradation were measured and are shown as a scatter plot (D). (E–H) MDA-MB-231 cells were transfected with PRKD2 siRNA or a dsRNA with a nontargeting, irrelevant sequence (Irr), together with pcDNA3.1 HisC-resPRKD2 (rescue PRKD2) or control pcDNA3 empty vector (−), as indicated (E and F). Hs578T and MDA-MB-435s cells were transfected with PRKD2 siRNA or a dsRNA with an irrelevant sequence (Irr), as indicated (G and H). Total lysates were subjected to blotting with the indicated antibodies (E and G). Activities of cell adhesion to collagen (Adhesion) and Matrigel chemoinvasion (Invasion) are shown (F and H). In F and H, data are presented as percentages calculated by normalizing the values obtained for the control cells as 100%. 4,213 ± 516 (∼4.21%), 2,562 ± 309 (∼2.56%), and 1,790 ± 271 (∼1.79%) control MDA-MB-231, Hs578T, and MDA-MB-435s cells, respectively, were calculated to have transmigrated per 6.4-mm-diam Matrigel-coated Boyden chamber filter under these conditions. Data are shown as mean ± SEM of triplicate experiments (error bars).
Article Snippet: cDNAs and transfection AMAP1 cDNA ( ) was cloned into pEGFP C1 (Takara Bio Inc.). cDNA for PRKD2, amplified by PCR from the first-strand cDNA of MDA-MB-231 cells, was cloned into pmVenus C1 (see below), pcDNA3.1 HisC (Invitrogen), or pCMV-V5 (see below).
Techniques: Immunoprecipitation, Multiple Displacement Amplification, Expressing, Cell Culture, Labeling, Staining, Transfection, Sequencing, Plasmid Preparation