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: Comparison of the expression levels of N -glycans and related glycosyltransferases between WT and WT/NaBu cells . The same amounts of cell lysates from WT and NaBu-induced K562 cells were subjected to 7.5% SDS-PAGE gel and stained with various lectins to assess glycosylation changes, such as E4-PHA ( A ), DSA ( B ), L4-PHA ( C ), LCA ( D ), MAA ( E ), and SNA ( F ) lectins, which preferentially recognizes the bisecting GlcNAc, GlcNAcβ1,4 mannosyl-, GlcNAcβ1,6 mannosyl-, α1,6 fucose, α2,3 sialylated, and α2,6 sialylated N -glycans, respectively. α-Tubulin was used as a loading control. G , expression levels of bisected N -glycans on the cell surface were detected by flow cytometry using E4-PHA lectin. H , mRNA levels of several N -acetylglucosaminyltransferases involved in the synthesis of GlcNAc branched N -glycans were determined using qPCR. GAPDH was used as an internal control, and values were normalized to WT cells without NaBu (set as 1.0). Statistical significance was assessed using the unpaired Student’s t test, with p values indicated as ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001, or denoted as no significance (ns). DSA, Datura stramonium agglutinin; E4-PHA, Phaseolus vulgaris erythroagglutinin, LCA, Lens culinaris agglutinin; MAA, M . amurensis ; NaBu, sodium butyrate; qPCR, quantitative PCR; SNA, S . nigra 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: Comparison, Expressing, SDS Page, Staining, Control, Flow Cytometry, Real-time Polymerase Chain Reaction
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