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. 2021 Sep 16;11:18435. doi: 10.1038/s41598-021-97777-4

Figure 1.

Figure 1

Disease mutations destabilize the regulatory protein MLC1 in multiple cell types. (A,B) The correlation between the PM density and cellular total protein expression of MLC1 variants (wt, P92S, C326R) was measured at 37 °C and upon temperature rescue (26 °C, 24 h) in HeLa cells by cs-ELISA and Western blot analysis with densitometry. (C) The PM turnover of MLC1 variants was monitored in the primary astrocytes, HeLa, and astrocytic U251N cells using cs-ELISA. (D) Internalization rates of the MLC1 variants (wt, P92S, C326R) were determined using cs-ELISA. Internalization was initiated at 37 °C for 4 min after anti-HA binding at 4 °C in HeLa cells maintained at 37 °C or after following low-temperature rescue (26 °C, 24 h). Statistical significance in the internalization rate of non-rescued (37 °C) and low-temperature rescued (26 °C) MLC1 variants are indicated on the right panel (U251N, 26 °C). (E) The endosomal recycling rates of MLC1 variants [wt, P92S (PS), C326R (CR)] were measured in HeLa and astrocytic U251N cells as described in “Materials and methods”. (F) Correlation between internalization rates (%/4 min) and relative cell surface densities (% of the wt) of eight MLC1 variants in HeLa cells at 37 °C (red squares) or after temperature folding rescue at 26 °C (24 h, blue squares). Data are from the Fig. S1B,C. Means ± SEM, n ≥ 3, *p < 0.05, **p < 0.01, ***p < 0.001.