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. 2023 Feb 25;26(4):106274. doi: 10.1016/j.isci.2023.106274

Figure 1.

Figure 1

Novel cysteine controls NRP1 stability

(A) Multiple alignment of the sequences of PAN domain of representative proteins from seven different organisms highlights the position of conserved cysteines.

(B) Schematic diagram of amino acid sequence represents NRP1 vestigial PAN domain along with the four marked cysteines (Cys82, Cys104, Cys147, and Cys173) and the subsequent mutant version where conserved cysteines were mutated to alanine (Ala82, Ala104, Ala147, and Ala173).

(C) Immunoblot analysis of whole-cell lysates derived from 293T cells, transfected with HA-NRP1 WT and different single cysteine mutants of HA-NRP1 and HA-NRP1-4Cys-4Ala constructs as indicated. 24 h post-transfection, cells were split, and cells were treated with 100 μg/mL cycloheximide (CHX) 20 h later. At the indicated time points, whole-cell lysates were prepared for immunoblot analysis. Representative image of n = 3 biological replicates.

(D) Quantification of the band intensities in (c). The intensities of HA-NRP1 (WT and mutants) bands were normalized to actin and then normalized to HA-NRP1 WT. Data are represented as mean ± SD, n = 3, and ∗, p < 0.05; ∗∗, p < 0.005; ∗∗∗, p < 0.0005. were calculated with one-way ANOVA.