Figure 3. Ecm29 KO neurons exhibit... | Rockefeller University Press

Figure 3.

$Ecm29 KO neurons exhibit aberrant NKCC1 accumulation. (A) Total NKCC1 levels increase in Ecm29−/− KO brain cortical lysates. (A1) Western blots of lysates showing NKCC1 and KCC2 protein expression at indicated developmental stages. Arrowhead denotes immunoreactive bands corresponding to NKCC1. (A2) Quantification of protein levels from all experiments performed as described in A1 at different time points and compared with corresponding levels at both E16 and P19 (top panel) or normalized to actin as an internal control (bottom panel). Data represent mean ± SEM from more than three independent experiments; *, P < 0.05, multiple t tests. rel., relative. (B1) Cell surface biotinylation assay of brain tissue lysates at indicated developmental stages. Representative Western blot of total lysate, streptavidin pull-down (surface-bound), and supernatant fractions probed with antibodies to Ecm29, NKCC1, KCC2, the inhibitory synapse markers gephyrin and Neuroligin-2 (NL2), and the AIS-associated factors NF-186 and GABAa α2 receptors, as indicated. Arrowhead denotes immunoreactive bands corresponding to NKCC1. (B2) Histogram showing quantitation of proteins (±SEM; n > 3 independent experiments; *, P < 0.05, multiple t tests for each factor) in the surface-bound fraction. (C) Ecm29−/− KO neurons exhibit altered NKCC1 accumulation at the AIS. (C1 and C2) Representative confocal images of 7-DIV neurons coimmunostained with AnkG and NKCC1 (C1) or Trim46 and KCC2 (C2) antibodies, as indicated. Rightmost panels show regions of interest (dashed boxes) of the AIS represented at higher magnification, with NKCC1 (C1) or KCC2 (C2) staining intensity indicated by a linear pseudocolor scale. Bar: 20 µm. (C3) Traces from experiments performed as described in (C1) and (C2), showing NKCC1 and KCC2 staining along the first 60-µm region of axonal (right panels) and dendritic (left panels) processes. Light blue rectangles mark AnkG/Trim46-(+) AIS regions. Data points represent signal intensities (±SEM, n = 75–92 neurons for each set of experiments; **, P < 0.01, t test). (D) NKCC1 half-life increases in Ecm29−/− KO cortical cultures. (D1 and D2) Paradigm and representative blots for pulse-chase experiments on AHA-labeled, newly synthesized proteins in 5-DIV cortical cultures of wild-type, Ecm29 KO, or Ecm29 KO transfected with plasmid encoding Ecm29ΔC-mCherry, followed by AHA signal detection (top panel, D2) and Western blotting (bottom panel, D2) of the IP sample with an NKCC1 antibody. Arrowhead denotes immunoreactive bands corresponding to NKCC1. (D3) Summary of AHA signal intensity normalized to that of the corresponding Western blot bands (±SEM, n = 3 independent experiments; **, P < 0.01, t test) at different time points as indicated. NKCC1 protein t1/2 was calculated by exponential decay.$

Ecm29 KO neurons exhibit aberrant NKCC1 accumulation. (A) Total NKCC1 levels increase in Ecm29^−/− KO brain cortical lysates. (A1) Western blots of lysates showing NKCC1 and KCC2 protein expression at indicated developmental stages. Arrowhead denotes immunoreactive bands corresponding to NKCC1. (A2) Quantification of protein levels from all experiments performed as described in A1 at different time points and compared with corresponding levels at both E16 and P19 (top panel) or normalized to actin as an internal control (bottom panel). Data represent mean ± SEM from more than three independent experiments; *, P < 0.05, multiple t tests. rel., relative. (B1) Cell surface biotinylation assay of brain tissue lysates at indicated developmental stages. Representative Western blot of total lysate, streptavidin pull-down (surface-bound), and supernatant fractions probed with antibodies to Ecm29, NKCC1, KCC2, the inhibitory synapse markers gephyrin and Neuroligin-2 (NL2), and the AIS-associated factors NF-186 and GABAa α2 receptors, as indicated. Arrowhead denotes immunoreactive bands corresponding to NKCC1. (B2) Histogram showing quantitation of proteins (±SEM; n > 3 independent experiments; *, P < 0.05, multiple t tests for each factor) in the surface-bound fraction. (C) Ecm29^−/− KO neurons exhibit altered NKCC1 accumulation at the AIS. (C1 and C2) Representative confocal images of 7-DIV neurons coimmunostained with AnkG and NKCC1 (C1) or Trim46 and KCC2 (C2) antibodies, as indicated. Rightmost panels show regions of interest (dashed boxes) of the AIS represented at higher magnification, with NKCC1 (C1) or KCC2 (C2) staining intensity indicated by a linear pseudocolor scale. Bar: 20 µm. (C3) Traces from experiments performed as described in (C1) and (C2), showing NKCC1 and KCC2 staining along the first 60-µm region of axonal (right panels) and dendritic (left panels) processes. Light blue rectangles mark AnkG/Trim46-(+) AIS regions. Data points represent signal intensities (±SEM, n = 75–92 neurons for each set of experiments; **, P < 0.01, t test). (D) NKCC1 half-life increases in Ecm29^−/− KO cortical cultures. (D1 and D2) Paradigm and representative blots for pulse-chase experiments on AHA-labeled, newly synthesized proteins in 5-DIV cortical cultures of wild-type, Ecm29 KO, or Ecm29 KO transfected with plasmid encoding Ecm29ΔC-mCherry, followed by AHA signal detection (top panel, D2) and Western blotting (bottom panel, D2) of the IP sample with an NKCC1 antibody. Arrowhead denotes immunoreactive bands corresponding to NKCC1. (D3) Summary of AHA signal intensity normalized to that of the corresponding Western blot bands (±SEM, n = 3 independent experiments; **, P < 0.01, t test) at different time points as indicated. NKCC1 protein t_1/2 was calculated by exponential decay.