18 amino acid secretion signal peptide  (Addgene inc)

Journal: Cells

Article Title: A Nanobody-Based Toolbox to Probe ApoE4 in the Secretory Pathway and Cytosol

doi: 10.3390/cells15050479

Figure Lengend Snippet: ER-targeted apoE nanobodies 9-74, 19-38, 17-69 and 18-91 bind apoE4 intracellularly and promote its intracellular retention in HEK293T cells. ( A ) Schematic representation of the ER-targeted nanobody construct containing an N-terminal mouse IgH secretion signal peptide (SEC2), a C-terminal HA-tag, and a KDEL ER retention sequence. ( B ) ApoE4 expression construct containing an N-terminal 18-amino-acid signal peptide (SEC1). ( C ) Schematic depiction of cells expressing apoE4 and an ER-targeted apoE nanobody. The KDEL-equipped nanobody is directed to the ER, allowing its interaction with apoE4 as it passes through the secretory pathway. ( D ) Microscopy images showing colocalization (yellow) of apoE4 (green) and ER-targeted nanobodies (Nb-KDEL; red) in HEK293T cells. Scale bar: 10 µm. ( E ) Scatter dot plot of the averaged Pearson’s correlation coefficient (PCC averaged ) values quantifying colocalization between apoE4 and each ER-targeted nanobody. Each dot represents the average PCC value of one independent experiment (~15 cells per experiment). Black horizontal lines and error bars indicate the mean ± standard deviation (SD; n = 3). All nanobodies displayed PCC averaged values significantly greater than 0.5 (red dotted line) and approaching 1, indicative of strong colocalization (Fisher Z-transformation followed by a one-sample t -test against a reference value of 0.5). * p < 0.05; ** p < 0.01; *** p < 0.001. ( F ) Co-IP assays showing interaction between each of the ER-targeted apoE nanobodies (Nb-KDEL) and apoE4 in cell extracts of co-transfected HEK293T cells (lanes 6). Co-IP was performed using anti-HA agarose beads and analyzed by Western blot. Two negative controls were included: NC1, a co-IP using the extract of cells transfected with apoE4 alone (lanes 4), and NC2, a co-IP using the extract of cells co-transfected with apoE4 and an ER-targeted GFP nanobody (lanes 5). For each condition, the corresponding input (cell extract) is shown (lanes 1–3). Uncropped blots are shown in . ( G ) Western blot analysis showing increased apoE4 levels in cell extracts (left) and reduced apoE4 levels in conditioned medium (right) from HEK293T cells co-transfected with apoE4 and each of the ER-targeted apoE nanobodies, compared to cells co-transfected with apoE4 and an ER-targeted GFP nanobody control. Actin was used as a loading control. Uncropped versions of the shown blots, together with replicate blots (n = 3), are provided in . ( H ) Quantification of intracellular apoE4 levels (cell extract Western blots). ApoE4 levels were normalized to actin and expressed relative to the GFP nanobody control within each blot/replicate. Individual data points are shown as dots (n = 3); bars represent mean ± SD. Raw data are provided in . ( I ) Quantification of secreted apoE4 levels (conditioned medium Western blots). ApoE4 levels were expressed relative to the GFP nanobody control within each blot/replicate. Individual data points are shown as dots (n = 3); bars represent mean ± SD. Raw data are provided in .

Article Snippet: The pCMV4-ApoE4 plasmid, encoding apoE4 with an N-terminal 18-amino-acid secretion signal peptide (SEC1), was obtained from Addgene (Watertown, MA, USA, cat. no. 87087).

Techniques: Construct, Sequencing, Expressing, Microscopy, Standard Deviation, Transformation Assay, Co-Immunoprecipitation Assay, Transfection, Western Blot, Control