JCS009001 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. No alteration in the distribution of different plasma membrane markers in lvsB mutant cells. Immunofluorescence analysis of WT and lvsB mutant cells with antibodies recognizing p25 (A), H36 (B) or PM4C4 (C) (red). Cells were also stained to detect p80 (green). Bars, 5 μm.

• Supplemental Figure S2 -

  Fig. S2. Strategy used to obtain npcA knockout cells. (A) A knockout construct was generated by cloning on each side of the blasticidin resistance cassette the 5ʼ and 3ʼ portions of the NPCA gene (position 295-1212 and 3100-4140 respectively) obtained by PCR amplification on Dictyostelium genomic DNA (using primers a to d). DH1-10 cells were then transfected with this linearized construct and blasticidin selection (10 μg/ml) was applied. Primers e and f were used in genotyping analysis using PCR amplification to identify the npcA knockout clones generated by a double recombination event. (B) These primers generated no amplicon in knockout cells, contrary to WT cells. To confirm this result, the same samples were also tested using an irrelevant primer pair to confirm the presence of genomic DNA in the samples. Arrows indicate expected PCR products.