Figure 5. Cer is delivered... | Rockefeller University Press

Figure 5.

$Cer is delivered intercellularly via lipoprotein. (A) Agarose gel of Xbp1 cDNA amplicons in AML12 treated with Ctrl/CM. Fractions with M.W. >100 kDa by filtration were also used. (B) Agarose gel of Xbp1 cDNA amplicons in AML12 treated with sEV and the remaining eluents isolated from Ctrl/CM using qEV column. (C) Transmission electron microscope images of sEV and the combined remaining eluents used in B. Scale bar, 100 nm. (D) FPLC of the remaining eluents used in B. A total of 40 fractions were collected, and the cholesterol concentrations in each fraction were determined. (E and F) Agarose gel of Xbp1 cDNA amplicons (E) and western blot of p-IRE1α, XBP1s, and BiP (F) in AML12 treated with each fraction from (D) or fraction combinations denoted as I-VI as shown in E. The diamond indicates the nonspecific band. (G) Western blot of p-IRE1α, XBP1s, p-PERK, and BiP (upper panel) and agarose gel of Xbp1 cDNA amplicons (lower panel) in AML12 treated with Ctrl/CM generated by culturing 3T3-L1 using DFBS, VLDL/LDL-containing FBS, HDL-containing FBS, or VLDL/LDL/HDL-containing FBS. The diamond indicates the nonspecific band. (H) Coomassie brilliant blue staining of 3T3-L1–derived Ctrl/CM treated with 100°C heating or 100 μg/ml PK. (I) Western blot of p-IRE1α, XBP1s, p-PERK, and BiP (upper) and agarose gel of Xbp1 cDNA amplicons (lower) in AML12 treated with regular, 100°C heated, or 100 μg/ml PK-pretreated Ctrl/CM. The diamond indicates the nonspecific band. PK, proteinase K; FPLC, fast-protein liquid chromatography. Source data are available for this figure: SourceData F5. Refer to the image caption for details.$

Cer is delivered intercellularly via lipoprotein. (A) Agarose gel of Xbp1 cDNA amplicons in AML12 treated with Ctrl/CM. Fractions with M.W. >100 kDa by filtration were also used. (B) Agarose gel of Xbp1 cDNA amplicons in AML12 treated with sEV and the remaining eluents isolated from Ctrl/CM using qEV column. (C) Transmission electron microscope images of sEV and the combined remaining eluents used in B. Scale bar, 100 nm. (D) FPLC of the remaining eluents used in B. A total of 40 fractions were collected, and the cholesterol concentrations in each fraction were determined. (E and F) Agarose gel of Xbp1 cDNA amplicons (E) and western blot of p-IRE1α, XBP1s, and BiP (F) in AML12 treated with each fraction from (D) or fraction combinations denoted as I-VI as shown in E. The diamond indicates the nonspecific band. (G) Western blot of p-IRE1α, XBP1s, p-PERK, and BiP (upper panel) and agarose gel of Xbp1 cDNA amplicons (lower panel) in AML12 treated with Ctrl/CM generated by culturing 3T3-L1 using DFBS, VLDL/LDL-containing FBS, HDL-containing FBS, or VLDL/LDL/HDL-containing FBS. The diamond indicates the nonspecific band. (H) Coomassie brilliant blue staining of 3T3-L1–derived Ctrl/CM treated with 100°C heating or 100 μg/ml PK. (I) Western blot of p-IRE1α, XBP1s, p-PERK, and BiP (upper) and agarose gel of Xbp1 cDNA amplicons (lower) in AML12 treated with regular, 100°C heated, or 100 μg/ml PK-pretreated Ctrl/CM. The diamond indicates the nonspecific band. PK, proteinase K; FPLC, fast-protein liquid chromatography. Source data are available for this figure: SourceData F5.