Journal: The Journal of Biological Chemistry
Article Title: The acetylglucosaminyltransferase GnT-Ⅲ regulates erythroid differentiation through ERK/MAPK signaling
doi: 10.1016/j.jbc.2024.108010
Figure Lengend Snippet: Effects of GnT-Ⅲ on NaBu-induced erythroid differentiation . A , MGAT3 KO K562 cell line was established by using CRISPR/Cas9 technology. Equal amounts of proteins from WT and two MGAT3 KO cell lines, with or without NaBu treatment, were subjected to lectin blotting using E4-PHA. α-Tubulin was used as a loading control. B , coloration was compared between WT and MGAT3 KO cells after treatment with 1 mM NaBu for 96 h. C , mRNA expression levels of HBA and HBB in WT and two MGAT3 KO cell lines, with or without NaBu treatment, were compared using qPCR. GAPDH served as the internal control. All values were normalized to the GAPDH levels, with the ratio of WT without NaBu set as 1.0. Data are presented as the mean ± SD from three independent experiments. ∗∗∗ p < 0.001; ns, no significance. D , expression levels of CD235a protein on the cell surface were analyzed by flow cytometry. E , expression levels of CD71 were analyzed by Western blotting. α-Tubulin was used as a loading control. The results are presented as the mean ± SD from three independent experiments. ∗ p < 0.05 and ∗∗∗ p < 0.001. mRNA expression levels of GYPA ( F ) and TFRC ( G ) in WT and MGAT3 KO-1 cells, with or without NaBu treatment, were compared using qPCR. GAPDH served as the internal control. All values were normalized to the GAPDH levels, with the ratio of WT without NaBu set as 1.0. Data are presented as mean ± SD from three independent experiments using one-way ANOVA with Tukey’s post hoc analysis. ∗∗∗ p < 0.001; ns, no significance. H , equal amounts of proteins were pulled down with E4-PHA-agarose, and the samples or cell lysates were subjected to Western blotting with an anti-CD71 antibody. α-Tubulin was used as a loading control. The relative ratios of CD71 containing bisecting GlcNAc were normalized to the total CD71 levels at each point. Data were quantified using ImageJ software and derived from three independent experiments. All values reflect one-way ANOVA with Tukey’s post hoc analysis as the mean ± SD. ∗p < 0.05 and ∗∗∗ p < 0.001. I , WT and MGAT3 KO cells were cultured with or without 50 mM cycloheximide (CHX), a protein synthesis inhibitor, for indicated times (0, 4, 8, 12, and 24 h). Equal amounts of proteins were subjected to Western blotting with anti-CD71 antibodies. α-Tubulin was used as a loading control. Relative ratios were calculated based on the density of CD71 relative to α-Tubulin at each indicated time point, with the ratio at 0 h (without CHX) set as 1.0. Values represent mean ± SD from three independent experiments. ∗ p < 0.05; ∗∗∗ p < 0.001; and ns, no significance. J , CD71 protein expression in WT and MGAT3 KO cells was detected using anti-CD71 antibody ( green ). The plasma membrane was stained using WGA lectin ( red ). Detection was accomplished using a ZEISS LSM 900 confocal microscope a ZEISS LSM 900 confocal microscope cells. Scale bars represent 10 μm. E4-PHA, Phaseolus vulgaris erythroagglutinin; NaBu, sodium butyrate; qPCR, quantitative PCR; WGA, Wheat germ agglutinin.
Article Snippet: The experiments were performed using the following antibodies and reagents: Antibodies against CD71 (13113), c-Kit (3074), p44/42 MAPK (ERK1/2; 9102), phospho-p44/42 MAPK (Thr202/Tyr204) (p-ERK1/2; 4370), SAPK/JNK (9252), phospho-SAPK/JNK (Thr183/Tyr185) (4668), p38 MAPK (9212), phospho-p38 MAPK (Thr180/Tyr182) (4511), the peroxidase-conjugated secondary antibody against rabbit (7074S), and U0126 (9903) were purchased from Cell Signaling Technology; biotinylated E4-PHA (B-1385), DSA (B-1185), biotinylated L4-PHA (B-1115–2), biotinylated SNA (B-1305), biotinylated Wheat germ agglutinin (B-1025-5), and ABC kit (PK-4000) were from Vector Laboratories; biotinylated LCA (J207) and E4-PHA agarose (J311) were obtained from J-Oil Mills; biotinylated MAM lectin (#BA-s7801-2) was purchased from EY Laboratories; The anti-α-Tubulin (T6199) antibody and peroxidase-conjugated secondary agents against mouse (AP124P), NaBu (303410), SAHA (HY-10221), and hemin (HY-19424) were acquired from Sigma; The antibody against CD235a (JC159) was from Thermo Fisher Scientific; The goat anti-mouse IgG Alexa Fluor 568, goat anti-rabbit IgG Alexa Fluor 488 and streptavidin conjugate Alexa Fluor 647 antibody were from Invitrogen; phosphotyrosine (p-Tyr) antibody (sc-7020) was from Santa Cruz; PNGase F was from Roche.
Techniques: CRISPR, Control, Expressing, Flow Cytometry, Western Blot, Software, Derivative Assay, Cell Culture, Membrane, Staining, Microscopy, Real-time Polymerase Chain Reaction
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