Fig 1: ALKBH5 enhances Ccl28 mRNA stability in a m6A-dependent manner.a RNA level of Ccl28 in IRI-Alkbh5fl/flKspCre group and IRI-Alkbh5fl/fl group was examined by RT-qPCR. n = 3 biologically independent animals. b Protein level of CCL28 in IRI-Alkbh5fl/flKspCre group and IRI-Alkbh5fl/fl group was examined by western blot. c Representative IHC staining data of CCL28 in kidney biopsies from IRI mice. (Magnification: ×400, Scale bar: 40 μm). d Serum CCL28 level was examined by ELISA. n = 5 biologically independent animals. e m6A modification of Ccl28 mRNA was detected by MeRIP-qPCR analysis using anti-IgG and anti-m6A antibodies. n = 3 biologically independent animals. f Relative enrichment of Ccl28 mRNA associated with ALKBH5 protein was identified by RIP assays using anti-IgG and anti-FLAG antibodies. n = 4 biologically independent experiments. g Relative activity of the pGL3-empty, pGL3-CDS, pGL3-5’UTR, and pGL3-3’UTR luciferase reporter in Ad-Control and Ad-ALKBH5 groups. n = 3 biologically independent experiments. h Relative activity of the pGL3-empty, pGL3-WT, pGLMut1, pGLMut2, pGLMut3, and pGLMut4 luciferase reporter in Ad-Control and Ad-ALKBH5 groups. n = 3 biologically independent experiments. i ALKBH5-silenced and control cells were treated with actinomycin D and harvested at 0, 2, 4, and 6 h. RNA decay rate was determined to estimate the stability of Ccl28 mRNA (normalized to the expression at 0 h). n = 3 biologically independent experiments. j RT-qPCR analysis of Ccl28 in mRTECs after different insulin-like growth factor 2 binding proteins (IGF2BPs) knockdown compared to NC treatment. n = 3 biologically independent experiments. k Western blot analysis of CCL28 after IGF2BP2 knockdown in H/R-treated mRTECs. Three biological repeated immunoblots have been performed. l RT-qPCR analysis of RIP assays in H/R-treated mRTECs showing the direct binding between the IGF2BP2 protein and Ccl28 mRNA. n = 3 biologically independent experiments. m ALKBH5-silenced with/without IGF2BP2-silenced cells were treated with actinomycin D and harvested at 0, 2, 4, and 6 h. RNA decay rate was determined to estimate the stability of Ccl28 mRNA (normalized to the expression at 0 h). n = 3 biologically independent experiments. Data are shown as means ± SD. Unpaired two-tailed Student’s t-test for (a), (d–h), (j), (l).