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. 2021 May 28;22(7):e52154. doi: 10.15252/embr.202052154

Figure EV5. Co‐localization of pS270 gephyrin and total gephyrin at synapses.

Figure EV5

  • A
    Co‐localization of total gephyrin (rbGPHN) and phosphorylated pS270 gephyrin clusters (mAb7a) in conventional epifluorescence images. This is a binarized version of the image shown in Fig 7A. Scale bar: 2 µm. After 4‐AP treatment, the percentage of rbGPHN clusters positive for mAb7a (binarized) showed only a minor reduction (middle panel, MW test, **P < 0.01), possibly as a result of decreased mAb7a immunoreactivity. The percentage of mAb7a clusters positive for rbGPHN was not changed (right panel, MW test P = 0.74). mean ± SD; TTX: n = 4,040 synapses, 4‐AP: n = 3,818, from two independent experiments.
  • B
    Gephyrin SSDs segmented from mAb7a clusters in dSTORM images did not show differences in size after 4‐AP treatment (KS test, P = 0.05, n = 810 synapses in TTX and n = 727 in 4‐AP conditions from three experiments).
  • C–E
    Variability of network activity in the control condition (CTRL, no treatment) in primary spinal cord neuron cultures. (C) The pooled results of two independent experiments (shown in D and E) show that the immunoreactivity of pS270 gephyrin (mAb7a antibody) is differentially modulated by pharmacological treatments that increase (4‐AP, red traces) or decrease (TTX, blue) network activity, relative to the control condition (CTRL, black). Some of these data (TTX and 4‐AP) are the same as the ones shown in Fig 7B and C. Number of synapses: n = 4,040 in TTX (blue traces), n = 3,818 in 4‐AP (red), n = 3,946 in the CTRL condition (black) from two independent experiments. (D, E) Separate analysis of the two experiments shows that while the difference between the TTX and 4‐AP conditions is consistent, the control can vary substantially between experiments. Number of synapses: n = 2,362 in CTRL, n = 2,577 in TTX and n = 2,283 in 4‐AP conditions in (D), and n = 1,584 in CTRL, n = 1,463 in TTX and n = 1,535 in 4‐AP in (E). KW test/Dunn's test, ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Source data are available online for this figure.