Skip to main content
. 2016 Nov 7;7:13365. doi: 10.1038/ncomms13365

Figure 7. PLA assay to confirm convergence of cellular signalling pathways on gephyrin in neurons.

Figure 7

(a) PLA signal for SUMO1 or SUMO2 conjugation on gephyrin in the presence or absence of His-PIAS3; (right panel) quantification of the PLA signal shows significant increase in SUMO1 and SUMO2 conjugation on gephyrin with co-expression of His-PIAS3. (b) PLA signal for SUMO1 or SUMO2 on eGFP–gephyrin, eGFP–S268A or eGFP–K148R/K724R mutants; (right panel) quantification shows significant reduction in SUMO1 and SUMO2 conjugation on eGFP–S268A or eGFP–K148R/K724R mutations. (c) eGFP signal and PLA signal for Ac-Lys on eGFP–K148R/K724R compared with eGFP–gephyrin; (right panel) quantification shows significant reduction of Ac-Lys conjugation on eGFP–K148R/K724R mutant. (d) PLA signal for Ac-Lys on eGFP–gephyrin, eGFP–S268A- and eGFP–gephyrin-transfected neurons treated with ERK1/2 inhibitor PD98059, and eGFP–gephyrin-transfected neurons with SUMO inhibitor 2-D08; (right panel) quantification of the Ac-Lys conjugation shows significant reduction when the ERK site is mutated or ERK pathway is pharmacologically blocked, or when SUMO conjugation on gephyrin is blocked. Images from three independent experimental replicates were analysed. Scale bar, 15 μm. Error bars are s.e.m.