Chronic exposure to paclitaxel diminishes phosphoinositide signaling by calpain-mediated neuronal calcium sensor-1 degradation -- Boehmerle et al. 104 (26): 11103 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

Boehmerle et al. 10.1073/pnas.0701546104.

Supporting Information

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SI Figure 5
SI Experimental Procedures
SI Figure 6
SI Figure 7

SI Figure 5

Fig. 5. Paclitaxel treatment does not alter P2Y or PI4K levels. Representative Western blots and the normalized immunoreactivity for P2Y2 (A) and P2Y4 (B) show unaltered P2Y2 and P2P4 levels after paclitaxel treatment (800 ng/ml; 6 h) (n = 5). NS, not significant.

SI Figure 6

Fig. 6. Paclitaxel treatment does not alter PI4K levels. (A) PI4K can be regulated by NCS-1. However, no alteration in protein levels is observed in paclitaxel-treated cells (800 ng/ml; 6 h) (n = 5). NS, not significant.

SI Figure 7

Fig. 7. Calcium is required for paclitaxel-induced degradation of NCS-1. Preloading cells with 1 mM of the cell-permeable Ca²⁺ buffer BAPTA/AM for 1 h abrogates NCS-1 degradation (n = 2).

SI Experimental Procedures

Immunoprecipitation and Western blot analysis

Antibodies used were as follows: anti-NCS-1 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-b actin and GAPDH (Abcam, Cambridge, MA), anti-P2Y2 and anti-P2Y4 (Alomone Labs, Jerusalem, Israel); anti-PI4K (Upstate, Temecula, CA). Blots were quantified by scanning densitometry using UN-SCAN-IT (Silk Scientific Corporation, Orem, UT) normalizing protein expression to the b-actin or GAPDH loading control.