roti®-blot 2  (Carl Roth GmbH)

Journal: PNAS Nexus

Article Title: APOE3 astrocytes can rescue lipid abnormalities and dystrophic neurites of APOE4 human neurons

doi: 10.1093/pnasnexus/pgag053

Figure Lengend Snippet: APOE3 astrocytes have higher levels of APOE and higher levels of large APOE particles at baseline and upon NPC1 inhibition. Three isogenic APOE3 and APOE4 hiPSC-derived neuron−astrocyte (iN + hAs) cocultures were maintained for 1 week and treated with either the Veh (PBS) or the NPC1-inhibitor U18666A (NPC1 (−); 10 µg/mL) for 3 days. A) Representative native PAGE followed by APOE western blot showing large and small APOE particles in the culture media. B) Quantification of large and small APOE particle band intensity from native PAGE blots. C) Total APOE levels measured in culture media using an APOE ELISA assay. D) Representative western blot showing total APOE levels in iN + hAs lysates. E) Quantification of APOE band intensity from western blots. APOE levels were normalized to total protein levels. Data for each condition were averaged from at least three independent experiments (full differentiation of all isogenic lines) per isogenic line in B and C, and from two independent experiments per isogenic line in E. Each data point represents an isogenic line in B, C, and E. Statistical analysis was performed using two-way ANOVA with pairwise matching. Significant differences ( P < 0.05) are indicated on the graphs.

Article Snippet: Media samples were mixed with Native Sample Buffer (Bio-Rad, #1610738) supplemented with G-250 Sample Additive (Invitrogen, #BN2004) and run on 4–20% polyacrylamide tris-glycine gels in the absence of sodium dodecyl sulfate, reducing agents or sample boiling, in tris-glycine buffer supplemented with 1:20 native PAGE Cathode Buffer Additive (Invitrogen, #BN2002), at 150 V for 15 min.

Techniques: Inhibition, Derivative Assay, Clear Native PAGE, Western Blot, Enzyme-linked Immunosorbent Assay