Journal: Journal of Translational Medicine

Article Title: CD24 associates with EGFR and supports EGF/EGFR signaling via RhoA in gastric cancer cells

doi: 10.1186/s12967-016-0787-y

Figure Lengend Snippet: Downregulation of CD24 alters the localization and degradation of EGFR. a The mRNA levels of CD24 and EGFR were detected by qPCR in SGC-7901 cells transfected with control siRNA or siCD24. b and c The level of EGFR on cellular membrane of SGC-7901 cells ( b ) and BGC-823 cells ( c ) was measured using flow cytometry. d Representative immunofluorescence images of SGC-7901 cells transfected with control siRNA or siCD24 staining for EGFR and phalliodin. Scale bar 10 μM. Asterisk P < 0.05 in the siCD24 cells relative to siRNA control cells

Article Snippet: Considering the importance of Src in regulating receptor internalization [ ], and CD24 has been reported to interact with Src [ ], in initial experiments, we treated SGC-7901 and BGC-823 cells with PP2 (Alexis Biochemicals), an Src inhibitor, and examined whether PP2 inhibited EGFR expression and CD24 association of EGFR in our experimental system.

Techniques: Transfection, Flow Cytometry, Immunofluorescence, Staining

Journal: Journal of Translational Medicine

Article Title: CD24 associates with EGFR and supports EGF/EGFR signaling via RhoA in gastric cancer cells

doi: 10.1186/s12967-016-0787-y

Figure Lengend Snippet: RhoA, but not Src, medicates EGFR expression by CD24 in SGC-7901 cells. a SGC-7901 and BGC-823 cells were treated with 20 μM PP2. 30 min later, and total protein extracts from cells were analyzed by Western blotting assays for CD24 and EGFR expression. b SGC-7901 cells were transfected with control siRNA or siCD24, and the activity of RhoA was measured. RhoA-GTP bands were quantified and normalized against RhoA level ( graph on the right). Asterisk P < 0.05 in the siCD24 cells relative to siRNA control cells. c SGC-7901 cells were transfected with control siRNA or siRhoA, and the expressions of RhoA and EGFR were measured. EGFR bands were quantified and normalized against GAPDH ( graph on the right). Asterisk P < 0.05 in the siCD24 cells relative to siRNA control cells. d SGC-7901 cells were transfected with GFP-tagged empty vector or RhoA-V14 plasmids, and the total cellular proteins were extracted and analyzed for expression of EGFR by Western blotting assays. Asterisk P < 0.05 in the CD24 overexpressing cells relative to control cells

Article Snippet: Considering the importance of Src in regulating receptor internalization [ ], and CD24 has been reported to interact with Src [ ], in initial experiments, we treated SGC-7901 and BGC-823 cells with PP2 (Alexis Biochemicals), an Src inhibitor, and examined whether PP2 inhibited EGFR expression and CD24 association of EGFR in our experimental system.

Techniques: Expressing, Western Blot, Transfection, Activity Assay, Plasmid Preparation

Journal: Journal of Translational Medicine

Article Title: CD24 associates with EGFR and supports EGF/EGFR signaling via RhoA in gastric cancer cells

doi: 10.1186/s12967-016-0787-y

Figure Lengend Snippet: Effects of EGF on EGFR expression and distribution in gastric cancer cells with knockdown of CD24. a SGC-7901 b BGC-823 and c AGS-1 cells transfected with control siRNA or siCD24 were in serum-free media overnight and incubated with EGF 20 ng/mL for indicated times, and then proteins extracted from the lysates were subjected to Western blotting assays to detect the expression of EGFR. GAPDH is used for control. d SGC-7901 and e AGS-1 cells transfected with control siRNA or siCD24 were in serum-free media overnight and incubated with EGF 20 ng/mL for 15 min, and representative microscopy images of SGC-7901 and AGS-1 cells stained for EGFR are shown. Cell nuclei were labeled with DAPI. Scale bar 10 μm. f SGC-7901 cells transfected with control siRNA or siCD24 were in serum-free media overnight. In addition to its protein synthesis blocked with cycloheximide (CHX, 10 μg/mL), the cells were then stimulated with EGF (20 ng/mL) for the indicated times. Then, the cells were lysed and EGFR level was determined by Western blotting assays. EGFR bands were quantified and normalized against GAPDH ( graph on the right). Asterisk P < 0.05 in the siCD24 cells relative to siRNA control cells

Article Snippet: Considering the importance of Src in regulating receptor internalization [ ], and CD24 has been reported to interact with Src [ ], in initial experiments, we treated SGC-7901 and BGC-823 cells with PP2 (Alexis Biochemicals), an Src inhibitor, and examined whether PP2 inhibited EGFR expression and CD24 association of EGFR in our experimental system.

Techniques: Expressing, Transfection, Incubation, Western Blot, Microscopy, Staining, Labeling

Journal: eLife

Article Title: Comment on ‘YcgC represents a new protein deacetylase family in prokaryotes’

doi: 10.7554/eLife.37798

Figure Lengend Snippet: ( A ) SDS-PAGE analysis of proteins used in this study. All proteins were expressed and purified as GST-fusion proteins. In terms of YcgC and YcgC S200A the GST-tag was removed by TEV protease during the purification steps. Staining of the gel was done by coomassie brilliant blue (CBB). For molecular masses see figure legend for . ( B ) Analytical size exclusion chromatography on a S200 10/300 GL column shows that YcgC WT and YcgC S200A as well as CobB and the corresponding catalytically inactive variant CobB H110Y display an almost identical elution profile. Moreover, RutR proteins show a nearly identical elution profile in analytical SEC runs indicating that RutR acetylation at K52 and K62 does not interfere with protein folding or its oligomeric state. ( C ) RutR-His 6 AcK52 and AcK62 are quantitatively acetylated. Shown are SDS-PAGE and immunoblot analyses of all RutR-His 6 proteins used in this study. Staining for AcK using an anti-acetyl-L-lysine antibody revealed a strong signal for RutR AcK52 and AcK62, whereas no signal was obtained for RutR WT. As loading control anti-His 6 staining was performed. ( D ) ESI-MS data show the quantitative and homogenous incorporation of acetyl-L-lysine into RutR. Shown is the deconvoluted spectrum on the true mass scale after software transformation yielding one single peak and the corresponding molecular mass as indicated. Expected mass non-acetylated RutR: 24567.5 Da; acetylated RutR: 24609.5 Da.

Article Snippet: After addition of 10 mM N-(ε)-acetyl-L-lysine (Chem-Impex International Inc.) and 20 mM nicotinamide (NAM) cells were grown for further 30 min before protein expression was induced by adding 300 µM IPTG.

Techniques: SDS Page, Purification, Staining, Size-exclusion Chromatography, Variant Assay, Western Blot, Software, Transformation Assay