INSR protein, human

Cells were washed in PBS and harvested in Tri-Reagent (Ambion, AM9738) by scraping. Total RNA was extracted and quantified (Nanodrop) before using 750 ng to synthesize cDNA (High Capacity cDNA Reverse Transcription Kit, Life Technologies, UK, 4368813). qPCR was performed on a 1/40 cDNA dilution using Taqman Assays-on-Demand (Applied Biosystems: CEBPA- Hs0029972_m1, CEBPB- Hs00942496_s1, PPARG- Hs01115729_m1, ADIPOQ- Hs00605917_m1, LEP- Hs00174877m1, INSR- Hs00961557_m1, GLUT4- Hs00168966_m1, FASN- Hs00188012_m1, ELOVL6- Hs00907564_m1, SCD- Hs00748952_m1, DGAT2- Hs01017541_m1, PNPLA2- Hs00386101_m1, PLIN1- Hs00160173_m1, LIPE- Hs00193510_m1) and Kapa Probe Fast Mastermix (Kapa Biosystems) on a QuantStudio 7 Flex (ABI). Triplicate reactions were performed in a final volume of 6 µL. Relative transcript expression was calculated using the ΔΔCt relative quantification method [26 (link)]. The ΔCt values of target genes were normalized to the ΔCt (geometric mean) of reference transcripts 18S, PGK1, PPIA and UBC.

The entire HPT and 80 mg of mWAT were each homogenized in 1 mL of QIAzol^® Lysis Reagent (Qiagen, Barcelona, Spain) using a tissue homogenizer (Polytron™ PT 2100, KINEMATICA^®) according to the corresponding manufacturers’ protocol (RNeasy Lipid Tissue Mini Kit—Qiagen) to obtain the total RNA from each sample. Quantification and quality (260/280 and 260/230 contamination ratios) of the RNA obtained were analyzed using a NanoDrop™ 1000 Spectrophotometer (Thermo Fisher Scientific, Madrid, Spain). RNA integrity was checked by agarose gel electrophoresis. The cDNA was synthesized from 2 µg/mL of total RNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Madrid, Spain) and the following RT-PCR program: 25 °C for 10 min, 37 °C for 120 min, 85 °C for 5 min and stored at 4 °C. The resulting cDNA was amplified by real-time PCR with TaqMan^® methodology in the iCycler MyiQTM Single Color Real-Time PCR Detection System (Bio-Rad Laboratories, Madrid, Spain). Specific Gene TaqMan Assays for Lepr, Insr, Pomc, Cartpt, Npy, Agrp, Lep, Adipoq, and Hprt as the housekeeping control gene, were obtained from Applied Biosystems. The Real-Time PCR program was set as follows: 95 °C for 10 min, 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Each PCR was performed in duplicate. The relative expression of each mRNA was calculated using the 2^−ΔΔCt method [28 (link)].
The TaqMan^® Gene Expression Assays used to amplify the different genes were: Rn01433205_m1 (Lepr); Rn00690703_m1 (Insr); Rn00595020_m1 (Pomc); Rn00567382_m1 (Cartpt); Rn00561681_m1 (Npy); Rn01431702_g1 (Agrp); Rn01433205_m1 (Lepr); Rn00565158_m1 (Lep); Rn00595250_m1 (Adipoq); and Rn01527840_m1 (Hprt1).

The DNA sequences encoding human TMD fragments of IGF1R (residues 931–962; IGF1Rtm), InsR (residues 952–982; InsRtm), and IRR (residues 916–948; IRRtm) were synthesized from six overlapping synthetic oligonucleotides by PCR and then cloned into pGEMEX-1 vector. The genes were under the control of the T7 promoter. The final construct of IGF1Rtm was with the leading methionine: M-N⁹³²FIHLIIALPVAVLLIVGGLVIMLYVFHRKR⁹⁶². InsRtm and IRRtm gene constructions contained N-terminal His-tag connected with target proteins by flexible GS linker MHHHHHHG-S⁹⁵²NIAKIIIGPLIFVFLFSVVIGSIYLFLRKR⁹⁸² and MHHHHHHGS-A⁹¹⁷GGLHVLLTATPVGLTLLIVLAALGFFYGKKR⁹⁴⁸, respectively. We use continuous-exchange cell-free expression (CECF) system based on E. coli S30 extract. The plasmids were used as DNA templates in the reactions [51 (link)] to produce the target proteins. The CECF production in the precipitate-CF mode was optimized with respect to concentrations of plasmid DNA using SDS-PAGE [28 (link),52 (link)]. The yield of synthesized TMDs was ~1.5 mg from 1 mL of RM. The ¹³C,¹⁵N-labeled algal amino acid mixture was used for production of isotopic labeled proteins. The reactions were incubated with gentle shaking overnight at 34 °C. The peptide precipitate was separated from the reaction mixture by centrifugation. The pellet was solubilized in a buffer containing 0.5% lauroyl sarcosine, 50 mM Tris/HCl, pH 8.0, 50 mM NaCl, and size exclusion chromatography was carried out using the Tricorn 10/300 Superdex 200 Increase column at 0.5 mL/min flow rate. Fractions containing the peptide were combined, then the peptide was precipitated by addition of TCA and washed with cold acetone three times. The pellet was solubilized in 1:1 (v/v) trifluoroethanol–water mixture and lyophilized. In order to incorporate TMDs into membrane-mimicking micelles or bicelles, the peptide powder was dissolved in 1:1 (v/v) trifluoroethanol–water mixture with the addition of n-dodecylphosphocholine-d38 (d38-DPC, 98%, CIL) at the peptide:detergent (P:D) molar ratio of 1:160 [28 (link)]. The mixtures were lyophilized overnight and re-dissolved in 270 μL of water buffer solution containing 25 mM sodium phosphate, 0.3 mM NaN₃, and 5% D₂O (v/v), pH 6.7.