rabbit polyclonal antibodies against fn1  (Proteintech)

Journal: Molecular Biosystems

Article Title: Proteomics profiling of interactome dynamics by colocalisation analysis (COLA) † †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6mb00701e Click here for additional data file. Click here for additional data file.

doi: 10.1039/c6mb00701e

Figure Lengend Snippet: Proteomics-based profiling of colocalisation (COLA) to reveal functional associations. (A) Outline of methodology. SILAC heavy or light labelled cells were subjected to subcellular fractionation then mixed with an equal amount of whole cell lysate from the opposite label for relative quantification. SILAC mixes were digested and analysed by LC-MS/MS. Averaged normalised SILAC ratios for all fractions together create a multi-variate localisation signature for each protein (GNAS was used as example). Signatures were then subjected to unsupervised hierarchical clustering with Euclidean average linkage. Bootstrapping was used to reveal clustering matches with high confidence (in color) from the rest (blacked out). GNAS, GNB1, and GNB2 which are known to constitute a complex are shown as example of a bootstrapped cluster. (B) Outline of the subcellular fractionations. Four independent fractionations procedures were used: (1) serial solubilisation of cellular proteins (fractions 1 to 6), (2) serial centrifugation combined with aqueous biphasic extraction which separates plasma membrane from internal membranes (fractions 7 to 10), (3) separation of actin-rich cellular protrusions using micro-porous transwell filters (fraction 11), (4) separation of the extracellular/secreted proteins by collecting conditioned media (fraction 12). (C) Validation of serial solubilisation method (fractions 1–6) by western blotting: PDI, ER membrane protein; DIS3, soluble nuclear protein; H2AX, nucleosome constituent; actin (ACT) and vimentin (VIM), cytoskeletal proteins; L = matching whole cell lysate control. (D) Validation of serial centrifugation method (fractions 7–10) by western blotting: N-cadherin (CHD2) and ezrin/radixin/moesin proteins (ERM), plasma membrane; early endosomal antigen-1 (EEA1), endosomal; PDI, ER membrane protein; H2AX, nucleosome constituent; L = matching whole cell lysate control. (E) Validation of protrusion purification method (fraction 11) by western blotting: VASP, protrusion; H2AX, nuclear; L = matching cell-body lysate control. (F) Validation of conditioned media collection method (fraction 12) by western blotting: fibronectin-1 (FN1), secreted; GAPDH, intracellular; L = matching whole cell lysate control. (G) Heat map of Pearson correlation coefficients between the two SILAC replicate series of fractionations with switched labelling. Cells were fractionated in duplicate with switching the labels. Collected fraction mixes have high similarity with their corresponding replicate, but low similarity with other fractions. (H) Plotted averaged Pearson's correlation coefficients within replicate fractions versus averaged Pearson's correlation coefficients between different fractions. While a high degree of similarity exists within replicate fractions suggestive of high reproducibility, similarity between different fractions is very low, indicating that each fraction is likely providing unique information. (I) Distribution of SILAC ratios for each fraction (averaged from two replicates). Red lines mark the standard deviations for each fraction. (J) Principle component analysis of subcellular fractions. PC1 and 2 represent over 50% of all data variation. No single fraction contributes towards the overall variation disproportionately. (K) Analysis of the reproducibility of the overall fractionation signatures between two biological replicates. Euclidean distances between each protein signatures from two reciprocally SILAC labelled COLA fractionations were calculated and plotted against each other, showing a highly significant correlation ( p < 1.0 × 10 –15 ). The Pearson correlation coefficient (CC) is displayed on the graph. (L) Graph of percentage of total bootstrapped clusters ( p < 0.05) vs. the number of proteins per cluster. The majority of clusters are constituted of 2–4 proteins, yet very large clusters are still detectable by COLA.

Article Snippet: Rabbit polyclonal antibody against FN1 (sc-9068) was from Santa Cruz.

Techniques: Functional Assay, Fractionation, Liquid Chromatography with Mass Spectroscopy, Centrifugation, Western Blot, Purification

Journal: Oncotarget

Article Title: Fibrotic microenvironment promotes the metastatic seeding of tumor cells via activating the fibronectin 1/secreted phosphoprotein 1-integrin signaling

doi: 10.18632/oncotarget.10157

Figure Lengend Snippet: ( A , B ) FN1 but not SPP1 silencing in fibrotic lung-derived fibroblasts attenuated the ability of CM-FLF to chemoattract tumor cells. The fibrotic lung-derived fibroblasts were transfected with the indicated siRNAs for 48 hours, followed by refreshment with 1% FBS-supplemented RPMI 1640. The conditioned medium was harvested 24 hours later, and then applied to the lower chamber of transwell. ( C , D ) FN1 and SPP1 silencing in fibrotic lung-derived fibroblasts abrogated the anti-apoptosis activity of CM-FLF. The fibrotic lung-derived fibroblasts were transfected with the indicated siRNAs for 48 hours, followed by refreshment with serum-free RPMI 1640. The conditioned medium was harvested 24 hours later, and applied to incubation with Hepa1-6 (C) and 4T1 (D) for 36 hours before DAPI staining. iMAX, treatment with transfection reagent RNAiMAX. NC, transfection with negative control duplex for siRNAs. Data are derived from three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant.

Article Snippet: Sections were incubated at 4°C overnight with mouse monoclonal antibody against SPP1 (1:500, ZM-0174, ZSGB-BIO, China) or rabbit polyclonal antibody against GFP (1:200, LS-C154219, LSBio, WA, USA), FSP1 (1:400, ZA-0257, ZSGB-BIO, China) and FN1 (1:200, ZA-0106, ZSGB-BIO, China), then immunostained using ChemMate DAKO EnVision Detection Kit, Peroxidase/DAB, Rabbit/Mouse (DakoCytomation, Glostrup, Denmark).

Techniques: Derivative Assay, Transfection, Activity Assay, Incubation, Staining, Negative Control