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. 2021 May 26;69(9):2235–2251. doi: 10.1002/glia.24032

FIGURE 2.

FIGURE 2

Analysis of M1‐aquaporin‐4 (AQP4) mRNA levels, alternative splicing and posttranslational control of Aqp4 gene in M23‐null mice spinal cord. (a) Reference sequence details of mouse AQP4 transcripts and schematic position of total‐ and M1‐AQP4‐mRNA‐specific primers used in qPCR. Sequences obtained by NCBI (Gene ID: 11829). (b) AQP4‐Isoform specific qPCR analysis in spinal cord of WT and M23‐null mice. No differences in M1‐AQP4 mRNA or AQP4‐tot mRNAs levels were observed between WT and M23‐null. n = 4 for each genotype. n.s. not statistically significant M23‐nullvs WT. Student's t test for unpaired data. (c) AQP4‐Δ4 specific RT‐PCR in WT and M23‐null mice. No trace of Δ4 isoform (218 bp) was observed in spinal cord, only the full‐length mRNA (299 bp) was detected. n = 4 for each genotype. (c) Analysis of AQP4‐protein degradation in spinal cord of WT and M23‐null mice. Early‐stopped SDS‐PAGE and Western blotting showed no sign of AQP4 degradation in M23‐null mice. n = 4 for each genotype. (e) Analysis of M1M23I protein stability in transfected HEK cells. Cycloheximide (CHX)‐treated M1 and M1M23I expressing cells were analyzed for M1‐AQP4 protein expression at 0, 4, and 8 h after CHX treatment as indicated in each lane. (f) Densitometric analysis of Western blotting reported in Panel (e). No difference in M1‐AQP4 protein posttranslational stability was observed between WT (M1‐AQP4) and mutated (M1M23I) protein. n = 3 for each time point, n.s. not statistically significant M1M23I versus M1 for each time point. Student's t test for unpaired data. (g) Analysis of M1M23I protein expression in the presence of M23‐AQP4 isoform in co‐transfected HEK cells. HEK cells were co‐transfected with five parts of M23‐AQP4 and with one part of M1M23I, to mimic the isoform ratio observed in WT spinal cord. The empty vector was used as control DNA to equalize the DNA amount. No sign of M1M23I protein stabilization was observed in the presence of M23‐AQP4 isoform. n = 4–6 for each condition. Student's t test for unpaired data. (h) Cycloheximide (CHX) assay analysis of M1‐AQP4 posttranslational stability in WT and M23‐null astrocyte primary culture. WT and M23‐null astrocyte were treated with 30 μM CHX for 0, 4, and 8 h and M1‐AQP4 was measured by Western blotting. No difference in the M1‐AQP4 stability was observed between WT and M23‐null astrocyte. n = 3 for each condition. Student's t test for unpaired data [Color figure can be viewed at wileyonlinelibrary.com]