Protein Kinase C{delta} and Calmodulin Regulate Epidermal Growth Factor Receptor Recycling from Early Endosomes through Arp2/3 Complex and Cortactin -- Lladó et al. 19 (1): 17 Data Supplement - Supplemental Materials -- Molecular Biology of the Cell

Protein Kinase C ${delta}$ and Calmodulin Regulate Epidermal Growth Factor Receptor Recycling from Early Endosomes through Arp2/3 Complex and Cortactin
Mol. Biol. Cell Lladó et al. 19: 17

Supplemental Materials

This article contains the following supporting material:

Figure S1 - Analysis of actin binding proteins localization upon CaM inhibition. NRK or COS1 cells were treated with W13 (5 µg/ml) for 60 min in the presence of EGF (100 ng/ml) or transferrin-TRITC, with or without rottlerin (5 µM) for the last 15 min at 37°C and fixed. (A) COS-1 cells were double-labelled with anti-annexin A1 and anti-EGFR. (B) Labelling with anti-annexin A2 and transferrin-TRITC in NRK cells is shown. (C) α-actinin was detected with specific antibody followed by Alexa Fluor 488 in NRK cells. (D) Immunofluorescence was carried out in COS-1 cells with anti-MARCKS. Scale bars, 10 µm.
Figure S2 - GFP-N-WASP ΔGBD mimics the results obtained with GFP-WA domain of N-WASP. COS1 cells transiently expressing GFP-N-WASP ΔGBD (N-WASP lacking the GTPase-binding domain) were incubated with W13 (5 µg/ml, 90 min) and EGF-TRITC (100 ng/ml, 15 min) at 37ºC and fixed. Scale bar 10 µm.
Table S1 - Quantification of cytochalasin D effects on EGF trafficking. After internalization of ¹²⁵I-EGF (5 ng/ml, 7 min), HeLa cells were incubated in the presence of W13 (7.5 µg/ml) ± cytochalasin D (0.1 µM) for 30 min at 37ºC to measure recycling (A) and degradation (B). The table shows the means of the % of inhibition after each treatment. Data were obtained from three independent experiments (±SD). *, p 0.05 and **, p 0.01 for Student's t test, indicating statistical significance of difference between each condition and its corresponding control.
Table S2 - EGF recycling is regulated by Arp2/3 but not by N-WASP. (A) Vero cells were transfected with GFP or with GFP-WA. 48 h after, cells internalized ¹²⁵I-EGF (5 ng/ml, 7 min) and were incubated in the presence of W13 (7.5 µg/ml) for 30 min at 37ºC to measure recycling. The table shows the means of four independent experiments (±SD). *, p 0.05 for Student's t test, indicating statistical significance of difference between the % of inhibition in each condition to its corresponding control. (B) After 48h of transfection with GFP or GFP-N-WASP ΔWA, Vero cells internalized ¹²⁵I-EGF (5 ng/ml, 7 min) and were treated with W13 (7.5 µg/ml) for 30 min at 37ºC to measure recycling. The means of five replicates from two independent experiments (±SD) are given. No statistical difference between each experimental condition and its control was found by Student's t test.