Figure 1.
NHERF1 and NHERF2 are expressed in human mast cells but they do not associate with C3aR.
(A) NHERF1 and NHERF2 expression was determined in human mast cell lines (HMC-1, LAD2) and CD34+ primary human mast cells (CD34+ MC) by RT-PCR. Transient transfectants were generated in HEK293 cells expressing HA-tagged C3aR or β2 adrenergic receptor (β2-AR) and (B) Flag-tagged NHERF1 or (C) NHERF2. Cells were exposed to buffer or C3a (100 nM, 37°C for 5 min) as indicated, lysed, immunoprecipitated with anti-HA-antibody, resolved by 10% SDS-PAGE, and transferred onto nitrocellulose membrane. Blots were then probed with anti-Flag antibody to detect NHERF binding to the receptor (top panel) or anti-HA antibody to examine receptor expression (middle panel). Western blotting was performed with anti-Flag antibody on the lysate samples (input) to determine NHERF expression levels (bottom panel). A representative blot from three independent experiments is shown.
Figure 2.
Knockdown of NHERF1 and NHERF2 in HMC-1 cells.
Cells were stably transduced with scrambled shRNA control lentivirus or shRNA lentivirus targeted against NHERF1 or NHERF2. Quantitative PCR was performed to assess (A) NHERF1 and (B) NHERF2 mRNA levels in shRNA control and NHERF knockdown (KD) cells. Results are expressed as a ratio of NHERF to GAPDH mRNA levels. Data represent the mean ± SEM from three independent experiments. Statistical significance was determined by unpaired two-tailed t test. ** indicates p<0.001. Western blotting was also performed to examine NHERF protein levels. Representative immunoblots of HMC-1 cells with knockdown of (C) NHERF1 and (D) NHERF2 from three different experiments are shown.
Figure 3.
Silencing NHERF1 or NHERF2 has no effect on agonist-induced C3aR desensitization.
(A) shRNA control, (B) NHERF1, and (C) NHERF2 KD HMC-1 cells were loaded with Indo-1(1 µM), stimulated with C3a (100 nM) for 5 min and intracellular Ca2+ mobilization was determined (solid lines). The cells were washed three times with ice-cold buffer, resuspended in warm buffer and exposed to a second stimulation of C3a (100 nM) and intracellular Ca2+ mobilization was again determined (broken lines). Representative traces from at least three separate experiments are shown.
Figure 4.
Knockdown of NHERF1 and NHERF2 does not affect C3aR internalization.
(A) shRNA control HMC-1 cells (B) NHERF1 knockdown (KD) and (C) NHERF2 KD cells were exposed to buffer or C3a (100 nM) for 5 min. Cells were washed with ice-cold FACS buffer, incubated with a mouse anti-C3aR antibody or an isotype control antibody followed by PE-labeled donkey anti-mouse IgG antibody and analyzed by flow cytometry. Representative histograms plots from an internalization experiment following exposure to buffer (red line) or C3a (blue line) in shRNA control and NHERF KD cells are shown. (D) shRNA control, NHERF1 KD and NHERF2 KD cells were exposed to C3a for different time periods and receptor internalization was determined as described above. Internalization is expressed as the percentage loss of C3aR following exposure to C3a. Data represent the mean ± SEM from three experiments.
Figure 5.
Knockdown of NHERF1 and NHERF2 does not affect C3a-induced ERK/Akt phosphorylation, or chemotaxis.
(A) shRNA control and NHERF KD HMC-1 cells were stimulated with C3a (100 nM) for indicated time intervals. Cell lysates were separated on SDS-PAGE and blots were probed with anti-phospho-ERK1/2 antibody followed by anti-rabbit IgG-HRP antibody. Immunoreactive band were visualized by SuperSignal West Femto maximum sensitivity substrate. The blots were also stripped and reprobed with anti-phopho-Akt and anti-ERK1/2 antibody followed by anti-rabbit IgG-HRP. A representative immunoblot from three similar experiments is shown. (B) shRNA control and NHERF KD HMC-1 cells were allowed to chemotax to C3a (10 nM, 37°C for 3 h). Data is represented as the total number of chemotaxed cells in the lower chamber. Bar graphs represent the mean ± SEM from three independent experiments.
Figure 6.
NHERF1 and NHERF2 contribute to C3a-induced degranulation response in LAD2 and CD34+ primary human mast cells.
LAD2 or CD34+ primary human mast cells were stably transduced with scrambled shRNA control lentivirus or shRNA lentivirus targeted against NHERF1 or NHERF2. Western blotting was performed to assess NHERF expression levels in shRNA control and NHERF KD (A) LAD2 and (C) CD34+ primary human mast cells. A representative blot from three independent experiments is shown. shRNA control or NHERF KD (B) LAD2 mast cells or (D) CD34+ mast cells were stimulated with indicated concentrations of C3a or CST (100 nM) and percent degranulation (β-hexosaminidase release) was determined. LAD2 cells were also stimulated by NP-specific IgE/NP-BSA (Ag) as a non-GPCR control. Data are mean ± SEM of three experiments. Statistical significance was determined by two-way ANOVA with Bonferroni's post test. ** indicates p<0.001.
Figure 7.
NHERF1 and NHERF2 contribute to C3a-induced NF-κB activation and chemokine CCL4 generation.
shRNA control, NHERF1 KD or NHERF2 KD HMC-1 cells were transiently transfected with NF-κB luciferase reporter gene construct along with C3aR. (A) Cells were stimulated with C3a (100 nM for 6 hr) and NF-κB-dependent transcriptional activity was determined by luciferase activity assay. Data is presented as relative luciferase activity normalized to Renilla luciferase activity. (B) Control or NHERF KD cells were stimulated with C3a (100 nM for 6 h) and CCL4 production was determined from the supernatant by ELISA. Data shown are mean ± SEM of three experiments performed in triplicate. Statistical significance was determined by two-way ANOVA with Bonferroni's post test. ** indicates p<0.001.