Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2015 May 1;26(9):1711–1727. doi: 10.1091/mbc.E14-07-1221

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© 2015 Cruse et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

“ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.

PMC Copyright notice

FIGURE 2: — Silencing MS4A4 alters KIT internalization, recycling to the plasma membrane and signaling in LAD-2 cells. (A) Flow cytometry histogram showing surface KIT expression at day 7 postinfection with shMS4A4. (B) Flow cytometry histogram showing surface KIT expression after 16 h of SCF withdrawal. (C) As in B, but followed by stimulation with 100 ng/ml SCF for 1 h. Isotype, black; scramble, blue; shMS4A4, red). (D) Average flow cytometry data from SCF exposure experiments, Values are geometric mean fluorescence intensity (MFI; error bars, SEM; n = 3). (E) Average flow cytometry data calculated as percentage expression normalized to scramble control (error bars, SEM; n = 3). (F) Average flow cytometry data from KIT internalization assays after exposure to 100 ng/ml SCF. SCF was withdrawn for 16 h before experiment (n = 5, p = 0.0008, two-way ANOVA scramble vs. shMS4A4). *p < 0.05, Sidak's posttest. (G) KIT kinetics assay using SCF pulse chase with 100 ng/ml SCF. The x-axis is the time spent at 37°C during chase (n = 3). (H) KIT degradation assay using pulse chase with 100 ng/ml SCF in the presence of 10 μM cycloheximide and staining for surface and intracellular KIT. (I) As in F, after exposure to 10 ng/ml SCF. BFA was included to eliminate repopulation of surface KIT from newly synthesized pools (n = 4, p = 0.047, two-way ANOVA scramble vs. shMS4A4). *p < 0.05, Sidak's posttest. (J) KIT kinetics assay using pulse chase with 10 ng/ml SCF. The x-axis is the time spent at 37°C during chase (n = 3). *p < 0.05, Sidak's posttest. (K) KIT degradation assay by using pulse chase with 10 ng/ml SCF in the presence of 10 μM cycloheximide and staining for surface and intracellular KIT. (L) Immunoblots for pKIT Y^568/570 (Invitrogen antibody), pAKT S⁴⁷³, and pERK T²⁰²Y²⁰⁴ in scramble shRNA control and shMS4A4- treated LAD-2 cells for the indicated time after stimulation with 10 ng/ml SCF. (M) Analysis of fluorescence intensity (LI-COR system) of phosphorylated KIT (M), AKT (N), and ERK (O) after correction against loading control normalized to percentage of scramble control at 2 min (mean + SEM, n = 5). *p < 0.05, **p < 0.01, ***p < 0.001, ANOVA with Sidak's posttest.