PMC

Characterization of p280. Alper-p280 was used to western blot normal mouse serum (NMS), fetal bovine sera (FBS), concentrated Iscove's modified essential medium (IMEM), and culture medium conditioned by MDA.MB.231 cells (a). MDA.MB.231 conditioned media (sup) and recombinant filamin-A were resolved by SDS-PAGE followed by electrotransfer to nitrocellulose. Western blots were then probed using Alper-p280 (b). MDA.MB.231 culture supernatant was subjected to calpain cleavage for the indicated times. Proteins were resolved by SDS-PAGE, transferred to nitrocellulose, and then blotted using Alper-p280 (c). Normal human mammary epithelial cells (HMEC) were fixed, permeabilized, and stained using rhodamine-conjugated phalloidin (column 1, green), and filamin-A using either a commercially available anti-filamin-A antibody (top middle panel, red) or Alper-p280 (bottom middle panel, red). Far right hand panels are merged images. Scale bars = 20 μm (d). SKBR3 and MDA.MB.231 breast cancer cells were stained as described in (d). Scale bars = 20 μm (e,f). Lysosomes of MDA.MB.231 cells were labeled using lysotracker, fixed, and then permeabilized. Cells were stained with Alper-p280 followed by Alexa488-conjugated anti-mouse secondary antibody. Scale bars = 50 μm (g). All data are representative of three independent experiments.

Evaluation of filamin-A levels in human brain cell lines, brain tissue, and plasma samples. Alper p280 was used to western blot conditioned culture media of the indicated cell lines (a). Alper-p280 was used to evaluate cellular filamin-A (p250) levels from the indicated whole cell extracts. Actin western blot analysis was done as a control for equal loading (b). Immunohistochemical analysis on formalin-fixed paraffin-embedded sections of representative brain lesions using Alper-p280 antibody against filamin-A. Negative staining in normal brain tissue (1) and World Health Organization grade 2 oligodendroglioma (2); 2+ cytoplasmic (subcellular) staining in occasional neoplastic cells in grade 2 astrocytoma (3) and 3+ cytoplasmic (subcellular) staining in neoplastic cells in grade IV glioblastoma multiforme (4), (5), and (6). Magnification, ×400. Arrows, filamin-A (c). Box plot analysis representing levels of the 280-kDa form of filamin-A in plasma detected by the Alper-p280 antibody and measured by ELISA in plasma. Soluble filamin-A levels were determined by ELISA from plasma samples obtained from normal controls, patients with arthritis, astrocytomas, or glioblastoma (GBM). Soluble filamin-A plasma concentrations were determined by generating a standard curve using normal human plasma spiked with known amounts of recombinant human filamin-A. P-values were determined by comparison with controls by ANOVA. Data are representative of four independent experiments performed in triplicate. All analyses were done under blinded conditions (d). Threshold Chart (e).

Filamin-A protein levels are elevated in breast cancer cell lines, breast carcinoma tumor tissues, and in plasma collected from breast cancer patients. Alper-p280 was used to western blot conditioned culture media of the indicated cell lines (a). Filamin-A immunohistochemistry was done as described in the `Materials and Methods'. Two examples of Alper-p280 staining in normal breast, one negative (panel 1) and one positive (panel 2), are shown. Alper-p280 detected filamin-A in invasive breast carcinoma (panel 3). The immunohistochemical staining for filamin-A is granular and cytoplasmic (subcellular) (inset). Magnification, ×20 (b). Alper-p280 was used to localize filamin-A in ultra-thin sections of normal breast tissue (panel 1) and non-metastatic breast cancer (panel 2) using the immunogold labeling technique as described in the `Materials and Methods' followed by electron microscopy. Filamin-A was below the level of detection in normal breast tissue. In non-metastatic breast carcinoma, filamin-A was present in the extracellular matrix (arrows), and within the vacuolar space (inset). (c). Box plot analysis representing levels of the 280-kDa form of filamin-A in plasma detected by the Alper-p280 antibody and measured by ELISA in plasma. Soluble filamin-A levels were determined by ELISA in plasma samples from females without breast cancer (Ctrl), non-metastatic breast cancer (non-met), and metastatic breast cancer (met). Soluble filamin-A plasma concentrations were determined by generating a standard curve using normal human plasma spiked with known amounts of recombinant human filamin-A. P-values were determined by comparison with controls by ANOVA. Data are representative of four independent experiments performed in triplicate. All analyses were done under blinded conditions (d). ELISA analyses of soluble filamin-A levels in plasma from patients with ovarian serous cancer (stage II–IV, n = 20) were conducted as described in panel D (e). Threshold chart is described in the text (f). An ELISA assay was used to compare the reactivity of TI10 and Alper-p280 against plasma samples isolated from normal controls, patients with non-metastatic breast carcinoma, and patients with metastatic breast carcinoma. Five samples from each group were analyzed in duplicate. Data are mean ± SD.