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Fig. 1 | Journal of Experimental & Clinical Cancer Research

Fig. 1

From: Extracellular gamma-synuclein promotes tumor cell motility by activating β1 integrin-focal adhesion kinase signaling pathway and increasing matrix metalloproteinase-24, -2 protein secretion

Fig. 1

SNCG binds cell membrane and the binding form of SNCG on tumor cells was evaluated. a, Secreted SNCG levels positively correlate with intracellular SNCG levels in colorectal cancer (CRC) cell lines. SNCG levels in the conditioned medium (CM) were directly detected by the Sandwich ELISA (upper panel), and the corresponding concentrated CM and cell lysates were analyzed by Western blot (lower panel). HSP70 and GAPDH were respectively used as loading controls for CM and cell lysates. Representative results from three independent experiments were presented. b, Representative immunohistochemical staining for membrane SNCG (brown) with haematoxylin counterstain in human colon adenocarcinoma tissues. c-d, Immunofluorescent staining was performed as described under “Methods”. Both recombinant GST-SNCG and SNCG bound cell membranes in different CRC cells (c); Membrane-binding of exogenous SNCG in RKO cells was detected as early as five minutes after treatment and maintained at almost similar levels (d). The results shown are representative of at least six different fields observed in each experiment and of three similar independent experiments. e, Membrane binding form of SNCG was evaluated in HT29 cells. Cells were lysed and subfractionated as described in the Methods section. The cytosolic supernatant (lane 1), membrane subfractions (lanes 2-10) obtained by sequential washing twice with PBS (lanes 2-3), 5mM EGTA (lanes 4-5), 2 M NaCl (lanes 6-7), 1% Triton X-100 (lanes 8-9), and Triton X-100-resistant pellet (lane 10) were evaluated by Western blot using antibody against SNCG. Membrane protein Annexin A2 was used as a control. f, Comparison of cell membrane binding form of endogenous SNCG with exogenously added SNCG. Membrane subfractions from HT29 cells expressing endogenous SNCG (upper panel) and LOVO cells treated with exogenous SNCG protein (lower panel) were evaluated by specific antibody to SNCG. Membrane subfractions were successively washed twice with 5 mM EGTA (lanes 1-2), 2 M NaCl (lanes 3-4) and 1% Triton X-100 (lanes 5-6). Lane 7, Triton X-100-resistant pellet. g, Phase separation of hydrophilic and hydrophobic proteins. Membrane fraction was described under “Methods”. Aliquots of the aqueous (A) and detergent (T) phases were analyzed by Western blot analysis

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