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. 2016 May 12;291(28):14732–14746. doi: 10.1074/jbc.M116.716514

FIGURE 7.

FIGURE 7.

Generation and characterization of Rab5a+/− and Rab5b+/− of Leishmania. A, schematic representation of the generation of LdRab5a or LdRab5b gene knock-out constructs. B, genomic DNA was isolated from the indicated cloned cells, and DNA (35 ng) from respective cells was analyzed by quantitative PCR using LDRab5a- or LdRab5b-specific primers to validate Leishmania:WT, Leishmania:Rab5a+/−, and Leishmania:Rab5b+/−. Amplification of Ld-GAPDH by specific primers (forward, 5′-GAAGTACACGGTGGAGGCTG-3′; reverse, 5′-CGCTGATCACGACCTTCTTC-3′) was used as a control. Results were analyzed with the comparative Ct method (2−ΔΔCt) and are expressed as relative quantity of Rab5a or Rab5b. C, to determine the transcript levels of LdRab5a and LdRab5b, cDNA was prepared from indicated cells, and RT-PCR was carried out using gene-specific primers. GAPDH was used as control. D, to determine the correct integration of LdRab5a-Hyg or LdRab5b-Hyg targeting knock-out cassette to one of the alleles of the LdRab5a or LdRab5b gene, PCR was carried out using forward primer upstream to LdRab5a (forward, 5′-CATCGCAAGCGTCGGTGATGGGC-3′) or LdRab5b (forward, 5′-CCTGTGCAGCCGCGCGCATTG-3′) of the gene-targeting construct and reverse primer (reverse, 5′-CCGCAGGACATATCCACGCCCTC-3′) specific for the hygromycin cassette from genomic DNA isolated from indicated cell types. Results are representative of three independent experiments. E, LdRab5a+/− cells specifically impair the fluid phase HRP uptake. Leishmania:WT, Leishmania:Rab5a+/−, and Leishmania:Rab5b+/− were incubated with 1 mg/ml HRP for 1 h at 23 °C, and HRP uptake by respective cells was measured as described previously. Results are represented as mean ± S.D. of three independent experiments and expressed as relative percentage of HRP uptake by control cells, arbitrarily chosen as 100%. F, LdRab5b+/− cells specifically impair receptor-mediated uptake of Hb. Leishmania:WT, Leishmania:Rab5a+/−, and Leishmania:Rab5b+/− were incubated with 6 μg/ml biotinylated Hb for 2 h at 23 °C, and Hb uptake by respective cells was measured as described under “Experimental Procedures.” Results are represented as mean ± S.D. of three independent experiments and expressed as relative percentage of Hb uptake by control cells, arbitrarily chosen as 100%.