The β- and -isoforms of type I PIP5K regulate distinct stages of Ca²⁺ signaling in mast cells

JCS048124 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. (A) Expression and subcellular localization of ECFP-Inp51 compared to untransfected 2H3 cells detected by confocal microscopy. ECFP-Inp51 shows plasma membrane localization in Inp cells. Bar, 25 µm. (B) PI(4,5)P₂ levels at the PM in 2H3 vs. Inp cells before and after stimulation with thapsigargin (0.25 µM). Normalized average PLCδ-PH-dsRed fluorescence intensity at the plasma membrane of 4 cells each for Inp and 2H3 cells. Error bars indicate SEM, and p value in selected region is shown.

• Supplemental Figure S2 -

  Fig. S2. (A) Representative images showing FcεRI expression on BMMCs labeled with 1 µg/ml Alexa488-IgE for 1 hr at 37°C. (B) Transient expression of wt and mutant PIP5kinase-Iγ87 and −Iγ90 in 2H3 cells. EGFP-tagged wt and K188A mutant PIP5kinase-Iγ were imaged in fixed cells. HA-tagged wt PIP5kinase-Iγ was detected in fixed cells following permeabilization and labeling with anti-HA mAb and Alexa488-anti mouse IgG₁ antibody. Images were collected using a Leica SP2 TCS confocal microscope and scale bars show 20 µm.

• Supplemental Figure S3 -

  Fig. S3. Representative Ca²⁺ responses for wt and PIP5kinase-Iγ^−/− BMMCs stimulated with thapsigargin. Traces show Ca²⁺ responses for wt (purple) and PIP5kinase-Iγ^−/− BMMCs (orange) stimulated with 0.25 µM thapsigargin in the presence of extracellular Ca²⁺.

• Supplemental Figure S4 -

  Fig. S4. Degranulation responses for wt and PIP5kinase-Iγ^−/− BMMCs. Histograms represent average degranulation responses to 0.01 µg/mL DNP-BSA (Ag) from four independent experiments and 4 µM A23187 from one experiment in the presence and absence of 2 µM BIM. Error bars show SEM.