JCS02997 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Expression levels of Glo3p and Gcs1p in arf1-16 and arf1-17. Integrants of wild-type ARF1 (WT; NYY0-1), arf1-16 (NYY16-1) and arf1-17 (NYY17-1) cells were transformed with pYO324 (vector), pNY24-GLO3 (2 μ GLO3), pNY24-3HA-GLO3 (2 m HA-GLO3), pNY24-GCS1 (2 m GCS1), and pNY24-GCS1-3HA (2 μ GCS1-HA). (A) The transformants were grown to an early log phase at 23°C, and incubated at 35°C for 1 hour. Total cell lysates (60 μg) were prepared and analyzed by immunoblotting by using the anti-HA antibody. (B) The transformants were diluted to a final concentration of 0.5310⁷, and 10 μl of tenfold dilutions were spotted on MCD (–Trp) plates. The plates were incubated at the indicated temperatures for 2-3 days.

• Supplemental Figure 2 -

  Fig. S2. Suppression activity of ArfGAPs for other arf1 ts alleles. arf1-11 (NYY11-1), arf1-13 (NYY13-1), arf1-14 (NYY14-1), and arf1-18 (NYY18-1) alleles were transformed with pYO324 (vector), pNY24-GLO3 (2 μ GLO3), pNY24-GCS1 (2 m GCS1), pNY24-AGE1 (2 μ AGE1) and pNY24-AGE2 (2 m AGE2), and the transformants were grown as in Fig. 1A.

• Supplemental Figure 3 -

  Fig. S3. Immunoblotting of B42-HA-Glo3p, B42-HA-Gcs1p, LexA-Arf1p, LexA-arf1-16p, and LexA-arf1-17p to examine their expression levels. The transformants that were used in Fig. 3 were grown to an early log phase at 30°C, washed with H₂O, and further grown in MVGal (–Trp, –His) medium for 16 hours. Total cell lysates (60 μg) were prepared and analyzed by immunoblotting using the anti-HA monoclonal (upper panel) and anti-Arf1p (lower panel) antibodies. Asterisk indicates a nonspecific band.