Fig 1: Testing the specificity of the antibodies against the GRP78 and HSP70.SDS–PAGE and Western blot analysis showing the specificity of each antibody (76-E6: ab25192 from Abcam; MAb159 from Dr Parkash S Gill at USC; C92F3A-5: sc-66048 from Santa Cruz Biotechnology) to the recombinant GST-tagged FL human GRP78 (rGST-GRP78/HSPA5) and recombinant FL human HSP70 (rHSP70/HSPA1A, ab92415 from Abcam). Recombinant proteins were prepared from E. coli.
Fig 2: Knockdown of GRP78 or CD44 alters cell morphology, reduces cell attachment, and impedes cell spreading in MCF7-LR cells.(A) Left: Western blot analysis of WCLs prepared from MCF7-LR cells transfected with control siRNA (sictrl) or siRNAs targeting GRP78 coding sequence, si78(1), or 3′ untranslated region (3′-UTR), si78(2), using antibodies against GRP78 (MAb159) or HSP70 (C92F3A-5). GAPDH served as a loading control. Right: Quantification of relative levels of GRP78 and HSP70. Data represents mean ± SEM from three biological repeats. (B) Sequence comparison of GRP78 siRNA target sites on human GRP78 and HSP70 genes. (C) Bright-field micrographs showing the morphology of MCF7-LR cells transfected with sictrl or siRNA targeting GRP78 or CD44. siCD44(1) targets 3′-UTR and siCD44(2) targets the common coding sequence. Scale bar, 100 μm. (D) Epifluorescent micrographs showing the F-actin organization of MCF7-LR cells transfected with sictrl or siRNA targeting GRP78 or CD44. The arrows indicate the F-actin bundles. Red, F-actin labeled with rhodamine phalloidin; blue, nuclei stained by DAPI. Scale bar, 50 μm. (E) Cell attachment assay. MCF7-LR cells were transfected with sictrl or siRNA targeting GRP78 or CD44 for 60 h before re-seeding onto collagen I–coated culture plates for 1 h. Attached cells were visualized by crystal violet staining. Scale bar, 50 μm. (F) Quantification of relative levels of cell attachment described in panel (E). Crystal violet staining of adherent cells was dissolved in 100% methanol, and the OD was measured at 595 nm. Data represent mean ± SEM from three biological repeats. *P < 0.05, **P < 0.01 (t test). (G) Kinetic measurement of cell spreading area. MCF7-LR cells were transfected with sictrl or siRNA targeting GRP78 or CD44 for 60 h before re-seeding onto collagen I–coated culture plates for the indicated times. Cells were stained with rhodamine phalloidin and visualized by epifluorescent microscopy. Cell areas were quantified by the FIJI-Image J software, and the results were represented by the box and whisker plot. n, number of analyzed cells; h, hour. **P < 0.01, ***P < 0.001 (t test). (H) Data obtained from panel (G) were represented by mean ± SEM for each condition.
Supplier Page from Abcam for Recombinant Human Hsp70 protein