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. 2019 Apr 24;13:164. doi: 10.3389/fncel.2019.00164

Figure 7.

Figure 7

tPA is associated with Rabs proteins. (A) Timeline of experiments. (B) Representative z-stack images of co-transfected cortical neurons (DIV20) with pCMV_tPA-HaloTag® (TMR ligand, in red) and pCMV_GFP-Rab5 (in green), followed by ICCs raised against MAP2 (in blue). tPA-HaloTag® colocalizes with GFP-Rab5 (early endosome) in axons (MAP2–; frame 1) and in dendrites (MAP2+; frame 2) with Pearson's coefficients respectively, of r = 0.822 and r = 0.649. (C) Representative diagrams of the diagrams of the percentages of colocalization between tPA-HaloTag® and GFP-Rab5 in function of total tPA-HaloTag® puncta. There are 42% of colocalization in axons and 36% in dendrites (N = 3 and N = 8 neurons respectively, from 4 independent cultures, p = 0.414). (D) Representative images of co-transfected cortical neurons (at DIV20) with pCMV_tPA-HaloTag® (reveals by TMR ligand, in red) and pCMV_CFP-Rab7 (in cyan), followed by ICCs raised against MAP2 (in blue). tPA-HaloTag® colocalizes with CFP-Rab7 (late endosome and lysosome degradation) in axons (MAP2–; frame 3) and in dendrites (MAP2+; frame 4) with Pearson's coefficients respectively of r = 0.833 and r = 0.761). (E) Representative diagrams of the percentages of colocalization between tPA-HaloTag® and CFP-Rab7 in function of total tPA-HaloTag® puncta. There are 18% of colocalization in axons and 16% in dendrites (N = 3 and N = 10 neurons respectively, from 4 independent cultures and p = 0.735). (F) Representative z-stack images of co-transfected cortical neurons (at DIV 20) with pCMV_tPA-HaloTag® (TMR ligand, in red) and pCMV_GFP-Rab11 (in green), followed by ICCs raised against MAP2 (in blue). tPA-HaloTag® colocalizes with GFP-Rab11 (recycling endosome) in axons (MAP2–; frame 5) and in dendrites (MAP2+; frame 6) with Pearson's coefficients respectively, of r = 0.720 and r = 0.671. (G) Representative diagrams of the percentages of colocalization between Representative diagrams of the percentages of colocalization between tPA-HaloTag® and GFP-Rab11 in function of total tPA-HaloTag® puncta. There are 57% of colocalization in axons and 46% in dendrites (N = 6 and N = 7 neurons respectively, from 2 independent cultures and p = 0.253). For all images: scale Bar: 5 or 50 μm (whole neuron).