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. 2016 Jul 7;99(1):174–187. doi: 10.1016/j.ajhg.2016.05.028

Figure 4.

Figure 4

Transient Expression and Intracellular Localization of SEC61A1_FLAG Variants in Human Embryonic Kidney 293 Cells

(A) Western blot detection of transiently expressed wild-type and mutated SEC61A1_FLAG proteins and endogenously expressed SEC61A, SEC61B, and tubulin at 36 hr post-transfection.

(B) Quantitative image analysis of wild-type and mutated SEC61A1_FLAG proteins demonstrating decreased amounts of mutated proteins compared to the wild-type. Results represent means of fold change ± SD of the relative signal intensities of mutated proteins to the wild-type protein from three biological replicates. Signal intensities of SEC61A1_FLAG proteins were normalized to that of α-tubulin. Statistical significance was assessed using Student’s t test; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

(C) Quantitative image analysis of the endogenously expressed SEC61A and SEC61B demonstrating that overexpression of neither protein significantly affected relative amounts of the endogeneously expressed SEC61A1 and SEC61B transient expression of SEC61A1. Results represent means of fold change ± SD from three biological replicates of the relative signal intensities of SE61A over SEC61B in cells expressing individual mutated SEC61A1_FLAG protein and empty vector compare to that of cells expressing the wild-type protein. Signal intensities of SEC61A and SEC61B were normalized to that of α-tubulin. Statistical significance was assessed using Student’s t test.

(D) Immunofluorescence analysis showing that the wild-type SEC61A1_FLAG is present in a finely granular (subpanel A) or coarsely granular (subpanel D) structures. Co-staining of wt-SEC61A1_FLAG with Golgi matrix protein GM130 (subpanel B) and with Protein disulphide isomerase (PDI) (subpanel E), a marker of endoplasmic reticulum (ER), demonstrating absence of the wt-SEC61A1_FLAG in the Golgi (subpanel C) but presence in the ER (subpanel F). p.Val67Gly (subpanels G and J) and p.Thr185Ala (subpanels M and P) variants of SEC61A1_FLAG are present in a form of intracellular clumps that are more pronounced in the latter. Co-staining with GM130 (subpanels H and N) and PDI (subpanels K and Q) demonstrating localization of both mutant proteins in the Golgi (subpanels I and O) as well as in ER (subpanels L and R). The degree of SEC6A1A_FLAG colocalization with selected markers is demonstrated by the fluorescent signal overlap coefficient values that range from 0 to 1. The resulting overlap coefficient values are presented as the pseudo color which scale is shown in corresponding lookup table.