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. 2024 Oct 11;27(11):111156. doi: 10.1016/j.isci.2024.111156

Figure 4.

Figure 4

SNAP25 decreases surface KCC2 in neuro-2a cells

(A) Immunoblot showing total and surface KCC2 in neuro-2a cells co-transfected with KCC2 and scrambled (scrSNAP25) or SNAP25 shRNA (shSNAP25-1). The blot was probed with the antibodies indicated on the right. Summary graph (n = 5) showing corresponding (B) total and (C) surface KCC2 abundance normalized to cells transfected with KCC2+scrSNAP25. Statistical significance was determined using an unpaired t-test with Welch’s correction. Asterisks denote significance from KCC2+scrSNAP25.

(D) Representative images (single z-plane) showing surface (gray) and total (green) KCC2 in neuro-2a cells co-transfected with KCC2 and scrSNAP25 (n = 41), shSNAP25-1 (n = 44) or shSNAP25-2 (n = 45). Red fluorescence represents transfected cells. n values indicate individual cells from three independent coverslips. Scale bar, 10 μm.

(E) Summary graph for (D) representing surface/total KCC2 normalized to cells transfected with KCC2+scrSNAP25. Statistical significance was determined using Kruskal-Wallis test and Dunn’s multiple comparisons test.

(F) Immunoblot showing total and surface KCC2 in neuro-2a cells co-transfected with KCC2 and RFP or SNAP25-P2A-RFP. The blot was probed with the antibodies indicated on the right. Summary graph (n = 8) showing corresponding (G) total and (H) surface KCC2 abundance normalized to cells transfected with KCC2+RFP. Statistical significance was determined using Mann-Whitney test. Asterisks denote significance from KCC2+RFP.

(I) Representative images (maximum intensity projection) showing PLA puncta (KCC2-SNAP25 interaction) in neuro-2a cells transfected with full length (KCC2-FL) KCC2 cDNA, and treated with DMSO (n = 106), PMA (n = 141) or Gö6983 (n = 134) and (J) summary graph showing the corresponding PLA/total KCC2 intensity. n values indicate individual cells from four independent coverslips. Statistical significance was determined using Kruskal-Wallis test and Dunn’s multiple comparisons test. Scale bar, 10 μm.

(K) Summary graph and (L) representative images (sum projection) showing PLA puncta intensity (KCC2-SNAP25 interaction) in neuro-2a cells transfected with KCC2 and treated with DMSO (n = 72) or PMA for 24 h (n = 79), 6 h (n = 72) and 1 h (n = 79). n values indicate individual cells from three independent coverslips. Statistical significance was determined using Kruskal-Wallis test and Dunn’s multiple comparisons test. Asterisks denote significance from DMSO. Scale bar, 10 μm.

(M) Summary graph and (N) representative images (sum projection) of PLA puncta intensity (KCC2-SNAP25 interaction) in cultured neurons treated with DMSO (n = 26), Gö6983 (48h, n = 29) or PMA (1h, n = 35). n values indicate individual cells from three independent cultures. Statistical significance was determined using Kruskal-Wallis test and Dunn’s multiple comparisons test. Asterisks denote significance from DMSO. Scale bar, 10 μm. All graphs represent mean ± SEM. ns - not significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.