Abi 7700 sequence detector system

To measure the expression levels of Tgfb and ribosomal protein lateral stalk subunit P0 (36B4) genes, total RNA was extracted from the liver tissues using RNAiso (Takara, Tokyo, Japan) and cDNA was prepared with Moloney murine leukemia virus reverse transcriptase (Invitrogen, Carlsbad, CA, USA). cDNA was amplified with the ABI 7700 sequence‐detector system (Applied Biosystems, Foster City, CA, USA) using a set of primers and probes that corresponded to Tgfb and 36B4 (endogenous control)^{41 (link)}. To measure the expression levels of Asb13, Icam1, Jun, Mfge, Mogat1, Plk3, and Srgn genes, total RNA from the liver tissues (six per group) or cells was extracted using a RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions. cDNA was prepared using SuperScript III Reverse Transcriptase (Invitrogen) and qRT-PCR was performed on a StepOnePlus Real-Time PCR System (Applied Biosystems) with Power SYBR Green PCR Master Mix (Applied Biosystems). Gene expression levels were analyzed by ΔΔC_T method using Gapdh as an internal control. Primers referred from PrimerBank (https://pga.mgh.harvard.edu/primerbank/index.html)^{43 (link)} were summarized in Supplementary Table 8.

TRIzol was used to isolate the total RNA. Thereafter, 1 µg of total RNA was reverse transcribed into cDNA using the Toyobo reverse transcription system (Osaka, Japan). SYBR Green Premix (Takara, Otsu, Japan) was used to conduct quantitative real-time PCR (qRT-PCR) on an ABI 7700 Sequence Detector System (Applied Biosystems, USA). The expression of target genes was normalized to the expression of GAPDH. We used the 2^−ΔΔCT method to determine the fold-change at the target gene level. Additional file 1: Table S2 displays all the primers used.

Skin allografts were harvested, and total RNA was extracted with the RNeasy kit (Qiagen). The reverse transcription was performed using Multiscribe Reverse Transcriptase Enzyme (Applied Biosystems, Foster City, CA). Real-time PCR was performed using an ABI 7700 sequence detector system (Applied Biosystems). To measure mRNA levels, we normalized the expression of target genes to the housekeeping gene HPRT, and data were represented as relative expression of the target gene to the housekeeping gene. The primers were used as follows: IL-12p35 sense, 5-CACGCTACCTCCTCTTTTTG-3; IL-12p35 anti-sense, 5-CAGCAGTGCAGGAATAATGTT-3; IL-12p40 sense, 5-AAACCAGACCCGCCCAAGAAC-3; IL-12p40 anti-sense, 5-AAAAAGCCAACCAAGCAGAAGACAG-3; HPRT sense, 5-GGTTAAGCAGTACAGCCCCAAAAT-3; and HPRT anti-sense, 5 -ATAGGCACATAGTGCAAATCAAAAGTC-3.