Figure 8: ARHGAP36 is expressed in neuroblastoma cells and promotes aberrant activation of the Hedgehog pathway.

(a) An amount of 10 μg lysate of the indicated cell lines was immunoblotted with the indicated antibodies. ARHGAP36 is only present in NGP and CLB-GA neuroblastoma cells. The predominant isoform seems to be isoform 3, expected size 46 kDa. (b) Histogram displays the distribution of the relative abundance of all measured proteins (n=4448). ARHGAP36 and PKAC are within the same bin (pink). Bar plot shows the log 10 abundance of the indicated proteins relative to ARHGAP36, GAPDH (437-fold) and PKAC (0.95-fold). PKAC is the summed total of PRKACA and PRKACB. (c) NGP cell lysates were immunoprecipitated with an ARHGAP36 antibody or IgG rabbit control, and immunoblotted for ARHGAP36 or PKAC. 75% less lysate and eluate was run for ARHGAP36. (d) Confocal micrographs of NGP or CLB-GA cells transfected with GFP-Rab5-QL. Fixed cells were subjected to immunofluorescence using GFP, ARHGAP36 and PKAC antibodies. Scale bar, 5 μm. (e) Representative immunoblot of NGP cells treated with SMARTpool short interfering RNA (siRNA) against ARHGAP36 (si36) for 24 h. Cells were stimulated with 10 μM Forskolin and 100 μM IBMX for 30 min before collecting. Lysates were immunoblotted with the indicated antibodies. Lipo: reagent only control. NT1: non-targeting oligo control. Bands were densitometrically evaluated, normalized first to tubulin then to Lipo. Mean of five independent experiments±s.d. **P<0.01, Student’s T-test compared with NT1. (f) Representative immunoblot of CLB-GA cells treated with individual or combined siRNA oligos against ARHGAP36 for 48 h. Bands were densitometrically evaluated, normalized first to tubulin then to Lipo. Mean of four independent experiments±s.d. **P<0.01 or *P<0.05, Student’s T-test compared with NT1. (g) Confocal micrographs of NGP cells subjected to immunofluorescence using antibodies against ARHGAP36 and PKAC. Scale bar, 10 μm. (h) NIH3T3 cells transfected with the indicated CFP-tagged constructs for 15 h were serum starved for a further 48 h before collecting. Gli1 mRNA levels measured by quantitative PCR with reverse transcription were normalized to β-actin. Data shown as mean of three repeats±s.e.m., ***P<0.001, Student’s T-test compared with control (i) As in h, except cells were transfected with CFP-PKI.