Rameters, Lipoprotein Profiles, and 7-hydroxy-4-cholesten-3-one (C4) Plasma lipid parameters, lipoprotein profiles immediately after separation by fast-protein liquid chromatography, and C4 concentrations were determined as previously described [29,30]. 2.3. Analysis of Circulating FGF15 Concentrations WT and LAL-KO mice fed a WTD for two weeks have been fasted for six h and gavaged with 200 corn oil. Ninety minutes post-gavage, blood was collected, and plasma was isolated by centrifugation at 5200g for 7 min at 4 C. Plasma FGF15 concentrations were measured by ELISA according to the manufacturer’s YN968D1 Autophagy protocol (R D Systems, Minneapolis, MN) [31]. 2.4. RNA Isolation, Reverse Transcription, and Quantitative Real-Time PCR RNA was isolated from tissues harvested from 6 h-fasted WT and LAL-KO mice fed a WTD for 6 weeks. Two micrograms of RNA were reverse transcribed applying the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA, USA). Three microliters of diluted cDNA (1:50) and 1 of each and every forward and reverse primer (Supplementary Table S1) have been mixed with 5 QuantiFast SYBR Green master mix (Qiagen, Hilden, Germany). Samples had been analyzed in duplicate and normalized towards the expression of peptidylprolyl isomerase A (Ppia, also known as cyclophilin A) as a housekeeping gene. Real-time PCR was performed on a Roche LightCycler 480 (Roche Diagnostics, Palo Alto, CA, USA). Expression profiles had been calculated using the 2-Ct strategy. two.5. Western Blotting Evaluation Samples were lysed in RIPA buffer, and protein concentrations were quantitated (DCTM Protein assay, Bio-Rad Laboratories, Hercules, CA, USA). Lysates (40 protein) had been separated by SDS-PAGE and transferred onto PVDF membranes. Non-specific binding sites on the Exendin-4 In Vivo membranes were blocked for 1 h at space temperature (five remedy of milk powder or 1 BSA in washing buffer). For detection with the proteins of interest, we used polyclonal antibodies against pERK (#9106) and ERK (#4695) (both 1:1000; Cell Signaling Technology, Danvers, MA), CYP7A1 (ab65596), and TFEB (ab2636) (both 1:1000; Abcam, Cambridge, Uk). Polyclonal anti-rabbit calnexin (1:1000; Santa Cruz, Heidelberg, Germany), actin (1:10,000; Merck KGaA, Darmstadt, Germany), or HDAC1 (#2062, 1:1000; Cell Signaling Technology) had been utilised as loading controls. HRP-conjugated goat anti-rabbit (1:2500) and rabbit anti-mouse antibodies (1:500) (Dako, Glostrup, Denmark) were visualized by enhanced chemiluminescence detection on a ChemiDocTM MP imaging method (Bio-Rad Laboratories).Cells 2021, ten,four of2.six. Electron Microscopy The little intestine in LAL-KO mice accumulates excess lipids, specifically in the proximal part [6]. Freshly harvested duodena from chow diet-fed mice in the fed state have been immediately fixed in 2.five (wt/vol) glutaraldehyde and 2 (wt/vol) paraformaldehyde, buffered in 100 mM cacodylate buffer pH 7.4, and incubated at room temperature for 3 h. Post-fixation, samples were treated with two osmium tetroxide (diluted in 200 mM cacodylate buffer) for 2 h at area temperature. Immediately after washing for 2 h in 100 mM cacodylate buffer, the specimens had been dehydrated in a graded series of ethanol (50 , 70 , 80 , 96 , 100 p.a.), infiltrated with propylene oxide/TAAB (Agar Scientific, Essex, Excellent Britain) embedding resin (propylene oxide for 1 h at space temperature, propylene oxide/TAAB 1:1 for 3 h at room temperature, propylene oxide/TAAB 1:3 o/n at 4 C), lastly embedded in pure TAAB resin, and polymerized (two 1.five h, 48 C). S.
