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

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

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: Expression levels of bisected N -glycans and MGAT3 were significantly decreased after treatment with U0126 . A , equal amounts of proteins from K562 WT cells treated with or without U0126 for the indicated times were subjected to lectin blotting using E4-PHA. α-Tubulin was used as a loading control. B , mRNA levels of MGAT3 were measured using qPCR in WT and WT/NaBu cells treated with or without U0126. GAPDH served as the internal control. All values were normalized to the GAPDH levels, with the ratio of WT without U0126 set as 1.0. Data are presented as mean ± SD from three independent experiments. p values were calculated using one-way ANOVA with Tukey's post hoc analysis. ∗∗∗ p < 0.001. C , effects of U0126 on expression levels of bisected N -glycans in cell lysates using E4-PHA lectin blotting in WT and WT/NaBu cells. α-Tubulin was used as a loading control. D , effects of U0126 on expression levels of bisected N -glycans on the cell surface were analyzed using flow cytometry in WT and WT/NaBu cells. NaBu, sodium butyrate; qPCR, quantitative PCR.

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: Expressing, Control, Flow Cytometry, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: N -Glycosylation of ß4 Integrin Controls the Adhesion and Motility of Keratinocytes

doi: 10.1371/journal.pone.0027084

Figure Lengend Snippet: (A) Schematic diagram of the potential N -glycosylation sites (N X (S/T)) on the ß4 integrin subunit. The sites corresponding to the putative N -glycosylation sites on the ß4 integrin subunit (Asn 327 , Asn 491 , Asn 579 , Asn 617 , and Asn 695 ) are shown by flags. Numbers and boxes indicate the number of amino acid residue and the four intracellular fibronectin type III repeats, respectively. TM, transmembrane region. (B) Cell lysates from normal keratinocytes were immunoprecipitated using a control IgG or an anti-ß4 integrin Ab. Immunoprecipitates were run on a 6% SDS-polyacrylamide gel and probed with the biotinylated L 4 -PHA lectin (upper left panel) and E 4 -PHA lectin (upper right panel) or anti-ß4 and anti-α6 integrin Abs (lower panels). IB, immunoblot. The black and white arrowheads indicate the ß4 integrin and α6 integrin subunits, respectively. Ordinates indicate molecular sizes in kDa of marker proteins. (C) Cell morphology of the WT and ΔNß4 integrin-expressing keratinocytes during cell culture. (D) The cell-spreading area in A was calculated using computer software (Image J). Each bar represents the mean ± S.D. of triplicate assays. *p<0.001 (unpaired t-test vs.WT). (E) 20 µg of cell lysates from control lacZ- (lac), WTß4, ΔNß4 integrin-expressing keratinocytes were run on a 6% gel under reducing conditions, blotted onto a nitrocellulose membrane, and then probed with an anti-ß4 integrin Ab. (F) Cell lysates were immunoprecipitated using a polyclonal Ab against ß4 integrin. Immunoprecipitates were run on a 6% SDS-polyacrylamide gel and probed with the indicated biotinylated lectins or an anti-ß4 integrin Ab. (G) Cell surface expression levels of α3, α6, ß1 and ß4 integrin subunits of WT or ΔNß4 integrin-expressing keratinocytes were examined using FACS analysis. Prior to analysis, cells were incubated with either the indicated integrin Abs or control IgG, followed by incubation with Alexa Fluor conjugated secondary Abs, as described under “ .” (H) Immunoprecipitates from WT or ΔN keratinocytes with an anti-α6 integrin were probed with an anti-ß4 (upper panel) or ß1 integrin (lower panel) Ab.

Article Snippet: Biotinylated DSA, L 4 -PHA, E 4 -PHA, ConA, SSA and MAM lectins were from Seikagaku Biobusiness Corporation (Tokyo, Japan).

Techniques: Immunoprecipitation, Western Blot, Marker, Expressing, Cell Culture, Software, Incubation