polyclonal antibodies hmgn2  (Millipore)

Journal: Journal of Cellular and Molecular Medicine

Article Title: The regulatory effect of acetylation of HMGN2 and H3K27 on pyocyanin‐induced autophagy in macrophages by affecting Ulk1 transcription

doi: 10.1111/jcmm.16788

Figure Lengend Snippet: Primer sequences for RT‐qPCR.

Article Snippet: The rabbit polyclonal antibodies against HMGN2 (9437S), LC3B (3868S), p62 (8025S), acetylated lysine (9441S), H3 (4499S) and H3K27ac (8173T) were purchased from Cell Signaling Technology; the rabbit polyclonal antibody against p‐ULK1 (ab203207) was obtained from Abcam, while the rabbit anti‐β‐actin (AB0035) monoclonal antibody was from Abways, China, and the rabbit polyclonal antibody against ULK1 (A5149) was purchased from Bimake Corporation.

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: The regulatory effect of acetylation of HMGN2 and H3K27 on pyocyanin‐induced autophagy in macrophages by affecting Ulk1 transcription

doi: 10.1111/jcmm.16788

Figure Lengend Snippet: Sequences of primers for mutation of HMGN2.

Article Snippet: The rabbit polyclonal antibodies against HMGN2 (9437S), LC3B (3868S), p62 (8025S), acetylated lysine (9441S), H3 (4499S) and H3K27ac (8173T) were purchased from Cell Signaling Technology; the rabbit polyclonal antibody against p‐ULK1 (ab203207) was obtained from Abcam, while the rabbit anti‐β‐actin (AB0035) monoclonal antibody was from Abways, China, and the rabbit polyclonal antibody against ULK1 (A5149) was purchased from Bimake Corporation.

Techniques: Mutagenesis

Journal: Journal of Cellular and Molecular Medicine

Article Title: The regulatory effect of acetylation of HMGN2 and H3K27 on pyocyanin‐induced autophagy in macrophages by affecting Ulk1 transcription

doi: 10.1111/jcmm.16788

Figure Lengend Snippet: PYO up‐regulates HMGN2ac in macrophages. RAW 264.7 cells were treated with 50 μM PYO or DMSO for 6 h, and TSA (50 nM) was used as a positive control for HMGN2ac. ⁴⁹ (A) IP showing the acetylation of lysine (ACE‐lysine) with (B) densitometric analysis showing the relative expression normalized by that of the DMSO group. (C) IP showing the HMGN2 with (D) densitometric analysis showing the relative expression normalized by the DMSO group. (E) The LC/MS mass spectrometer displaying the acetylation modification sites of HMGN2, and the amino acid sequences of HMGN2 of different species were compared and the acetylation sites were marked. NLS: nuclear localization signal domain, NBD: nucleosome binding domain and RD: regulatory domain. PM and THP‐1 cells were treated both with 50 μM PYO and with DMSO for 6 h, (F) IP HMGN2 showing the ACE‐lysine with (G) the densitometric analysis showing relative expression normalized by DMSO group. Data are expressed as mean ± SD, * P < 0.05 compared with the DMSO group, n = 3

Article Snippet: The rabbit polyclonal antibodies against HMGN2 (9437S), LC3B (3868S), p62 (8025S), acetylated lysine (9441S), H3 (4499S) and H3K27ac (8173T) were purchased from Cell Signaling Technology; the rabbit polyclonal antibody against p‐ULK1 (ab203207) was obtained from Abcam, while the rabbit anti‐β‐actin (AB0035) monoclonal antibody was from Abways, China, and the rabbit polyclonal antibody against ULK1 (A5149) was purchased from Bimake Corporation.

Techniques: Positive Control, Expressing, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Modification, Binding Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: The regulatory effect of acetylation of HMGN2 and H3K27 on pyocyanin‐induced autophagy in macrophages by affecting Ulk1 transcription

doi: 10.1111/jcmm.16788

Figure Lengend Snippet: Effect of HMGN2ac on the PYO‐mediated autophagy in the RAW 264.7 cells. The WT and KO RAW 264.7 cells were treated with 50 μM PYO or DMSO for 6 h. (A) Western blot showing LC3B II protein in the RAW 264.7 cells with or without Hmgn2 −/− upon PYO (50 μM, 6 h) or DMSO treatment, and (B) densitometric analysis showing relative expression normalized to that of the DMSO group. (C) Confocal microscopy images displaying the amount of intracellular LC3B puncta (green fluorescence, 630x) with or without Hmgn2 −/− upon PYO (50 μM, 6 h) or DMSO treatment. The nucleus was stained by DAPI (blue fluorescence, 630x), scale bar = 5 μm. (D, F) KO RAW 264.7 cells were transfected, respectively, with the GFP, 2K‐R, 3K‐R, 5K‐R and WT HMGN2 plasmids using jetPRIME for 24 h and then incubated both with 50 μM PYO and with DMSO for 6 h. The Western blot analysis showing the LC3B II protein. (E, G) Densitometric analysis showing relative expression normalized to that of the GFP plasmid. Data are expressed as mean ± SD, * P < 0.05, and n.s indicates no statistical difference, n = 3

Article Snippet: The rabbit polyclonal antibodies against HMGN2 (9437S), LC3B (3868S), p62 (8025S), acetylated lysine (9441S), H3 (4499S) and H3K27ac (8173T) were purchased from Cell Signaling Technology; the rabbit polyclonal antibody against p‐ULK1 (ab203207) was obtained from Abcam, while the rabbit anti‐β‐actin (AB0035) monoclonal antibody was from Abways, China, and the rabbit polyclonal antibody against ULK1 (A5149) was purchased from Bimake Corporation.

Techniques: Western Blot, Expressing, Confocal Microscopy, Fluorescence, Staining, Transfection, Incubation, Plasmid Preparation

Journal: Journal of Cellular and Molecular Medicine

Article Title: The regulatory effect of acetylation of HMGN2 and H3K27 on pyocyanin‐induced autophagy in macrophages by affecting Ulk1 transcription

doi: 10.1111/jcmm.16788

Figure Lengend Snippet: HMGN2ac might have no interaction with H3K27ac. The KO RAW 264.7 cells were transfected, respectively, with GFP, 2K‐R, 3K‐R, 5K‐R and WT HMGN2 plasmids by using jetPRIME, for 24 h, and then incubated with both 50 μM PYO and DMSO for 6 h. The Western blot showing H3K27ac in RAW 264.7 cells treated both with DMSO (A) and with PYO (C), and (B, D) the densitometric analysis showing relative expression normalized by the GFP plasmid. (E) Western blot showing HMGN2 protein in the RAW 264.7 cells both with and without TSA (20 nM) upon PYO stimulation, and (F) densitometric analysis showing relative expression normalized to that of the DMSO group. (G) RT‐qPCR displaying the Hmgn2 mRNA in the RAW 264.7 cells treated with PYO (50 μM, 6 h), and DMSO is the control. (H‐I) Co‐IP showing the interaction of HMGN2 with H3K27ac. Data are expressed as mean ± SD, *** P < 0.001, and n.s indicates no statistical difference, n = 3

Article Snippet: The rabbit polyclonal antibodies against HMGN2 (9437S), LC3B (3868S), p62 (8025S), acetylated lysine (9441S), H3 (4499S) and H3K27ac (8173T) were purchased from Cell Signaling Technology; the rabbit polyclonal antibody against p‐ULK1 (ab203207) was obtained from Abcam, while the rabbit anti‐β‐actin (AB0035) monoclonal antibody was from Abways, China, and the rabbit polyclonal antibody against ULK1 (A5149) was purchased from Bimake Corporation.

Techniques: Transfection, Incubation, Western Blot, Expressing, Plasmid Preparation, Quantitative RT-PCR, Control, Co-Immunoprecipitation Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: The regulatory effect of acetylation of HMGN2 and H3K27 on pyocyanin‐induced autophagy in macrophages by affecting Ulk1 transcription

doi: 10.1111/jcmm.16788

Figure Lengend Snippet: HMGN2ac and H3K27ac regulate Ulk1 transcription during autophagy. Western blot showing p‐ULK1 and ULK1 in RAW 264.7 cells (A) and PM cells (C) treated with 50 μM PYO at indicated time‐points, and (B, D) densitometric analysis showing relative expression normalized by the DMSO group. (E) RT‐qPCR analysis showing the relative Ulk1 mRNA level in the RAW 264.7 cells treated both with and without TSA (20 nM) upon PYO (50 μM, 6 h) stimulation. (F) The KO RAW 264.7 cells were transfected, respectively, with GFP, 5K‐R and WT HMGN2 plasmids by using jetPRIME for 24 h and then incubated both with 50 μM PYO and with DMSO for 6 h. RT‐qPCR showing the relative Ulk1 mRNA. (G) The schematic diagram depicting primer amplification sites in ChIP‐qPCR. TSS, transcription start site; −855 to −692, area of promoter region for primer designing. (H) ChIP‐qPCR showing the H3K27ac recruitment at the Ulk1 promoter in the RAW 264.7 cells treated both with and without TSA (20 nM) upon PYO (50 μM, 6 h) stimulation. (I, J) The KO RAW 264.7 cells were transfected respectively with GFP, 5K‐R, and WT HMGN2 plasmids by using jetPRIME for 24 h, then the cells were treated both with 50 μM PYO and with DMSO for 6 h. The ChIP‐qPCR analysis showing the HMGN2ac recruitment at the Ulk1 gene promoter. Data are expressed as mean ±SD, * P < 0.05, ** P < 0.01 and *** P < 0.001, and n.s indicates no statistical difference, n = 3

Article Snippet: The rabbit polyclonal antibodies against HMGN2 (9437S), LC3B (3868S), p62 (8025S), acetylated lysine (9441S), H3 (4499S) and H3K27ac (8173T) were purchased from Cell Signaling Technology; the rabbit polyclonal antibody against p‐ULK1 (ab203207) was obtained from Abcam, while the rabbit anti‐β‐actin (AB0035) monoclonal antibody was from Abways, China, and the rabbit polyclonal antibody against ULK1 (A5149) was purchased from Bimake Corporation.

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Transfection, Incubation, Amplification, ChIP-qPCR

Journal: Acta biochimica Polonica

Article Title: Non-histone nuclear protein HMGN2 differently regulates the urothelium barrier function by altering expression of antimicrobial peptides and tight junction protein genes in UPEC J96-infected bladder epithelial cell monolayer.

doi: 10.18388/abp.2017_1622

Figure Lengend Snippet: Figure 1. HMGN2 facilitates AMPs gene expression in UPEC-infected BECs 5637. (A) Elevated HMGN2 expression in UPEC J96-infected mice bladder epithelia. Mice bladders were administrated with UPEC J96 (108 CFU) by transurethral catheterization. Then the immuno-fluorescence assay was applied using anti-HMGN2 antibody (α-HMGN2, red) and DAPI (blue). Confocal microscopy images were acquired. Bars: 50 μm. (B) Western blotting showing the knockdown efficiency of siRNA oligo targeting HMGN2 under UPEC J96 (MOI=100:1) infection. (C–H) mRNA levels of AMPs genes affected by HMGN2 knockdown in J96-in fected 5637 cells. (Results presented relative to GAPDH and expressed as the mean ± S.D. (n=3)).

Article Snippet: The following antibodies were used: rabbit polyclonal HMGN2 antibody (Cell Signalling Technology), mouse monoclonal GAPDH antibody (Beyotime Biotechnology), Rabbit monoclonal Occludin antibody (invitrogen, Thermofisher scientific), ZO-1 (invitrogen, Thermofisher scientific), E-cadherin (Cell Signalling Technology).

Techniques: Gene Expression, Infection, Expressing, Fluorescence, Confocal Microscopy, Western Blot, Knockdown

Journal: Acta biochimica Polonica

Article Title: Non-histone nuclear protein HMGN2 differently regulates the urothelium barrier function by altering expression of antimicrobial peptides and tight junction protein genes in UPEC J96-infected bladder epithelial cell monolayer.

doi: 10.18388/abp.2017_1622

Figure Lengend Snippet: Figure 2. HMGN2 down-regulates barrier function and inhibits TJs-associated gene expression in J96-infected BECs 5637 monolayer. (A) BECs 5637 confluent monolayer stably over-expressing HMGN2 was exposed to increasing doses of UPEC J96 (MOI=0, 10, 50, 100) or heat inactivated UPEC J96 (HI J96 MOI=100:1) for 2 hours or to TGF-β (2 ng/ml) for 24 hours respectively. The relative Dextran permeabil ity was calculated by dividing fluorescence density of HMGN2 overexpression group (pEx-HMGN2) by control group (pEx-NC). (B) West ern blotting showing the over-expression of GFP- HMGN2 (α-HMGN2, marked by single asterisk; double asterisk indicates endogenous HMGN2). (C–E) BECs 5637 transfected with si-HMGN2 or pEX-HMGN2 followed by 2-hour UPEC J96(MOI=100:1) infection. siRNA-NC or pEX-NC was used as control. (C–D) mRNA levels and (E) protein levels of TJs-associated genes after treatments (Results presented relative to GAPDH and expressed as the mean ± S.D. (n=3)).

Article Snippet: The following antibodies were used: rabbit polyclonal HMGN2 antibody (Cell Signalling Technology), mouse monoclonal GAPDH antibody (Beyotime Biotechnology), Rabbit monoclonal Occludin antibody (invitrogen, Thermofisher scientific), ZO-1 (invitrogen, Thermofisher scientific), E-cadherin (Cell Signalling Technology).

Techniques: Gene Expression, Infection, Stable Transfection, Expressing, Fluorescence, Over Expression, Control, Transfection

Journal: Acta biochimica Polonica

Article Title: Non-histone nuclear protein HMGN2 differently regulates the urothelium barrier function by altering expression of antimicrobial peptides and tight junction protein genes in UPEC J96-infected bladder epithelial cell monolayer.

doi: 10.18388/abp.2017_1622

Figure Lengend Snippet: Figure 4. HMGN2 overexpression enhanced the disruption of BECs 5637 monolayer TJ during J96 infection. BECs5637 stably expressing GFP-HMGN2 or vehicle control were exposed to increasing doses of UPEC J96 (MOI=10, 50, 100) for 2 hours in the apical chamber of transwell apparatus. 100 μl of the medium from lower chamber were taken for Flat colony counting. Results presenting relative colony number between HMGN2 overexpression (pEx-HMGN2) and vehicle control (pEx-NC) (A) and representative plates are shown (B) (Results expressed as the mean ± S.D. (n = 3)).

Article Snippet: The following antibodies were used: rabbit polyclonal HMGN2 antibody (Cell Signalling Technology), mouse monoclonal GAPDH antibody (Beyotime Biotechnology), Rabbit monoclonal Occludin antibody (invitrogen, Thermofisher scientific), ZO-1 (invitrogen, Thermofisher scientific), E-cadherin (Cell Signalling Technology).

Techniques: Over Expression, Disruption, Infection, Stable Transfection, Expressing, Control

Journal: Acta biochimica Polonica

Article Title: Non-histone nuclear protein HMGN2 differently regulates the urothelium barrier function by altering expression of antimicrobial peptides and tight junction protein genes in UPEC J96-infected bladder epithelial cell monolayer.

doi: 10.18388/abp.2017_1622

Figure Lengend Snippet: Figure 5. A schematic diagram depicting dual functions of HMGN2 on the transcellular and paracellular route of UPEC invasion in Urothelium. LPS and other bacterial toxins activate HMGN2 expression, which in turn elevates expression of general innate immune response genes, such as IL-8, IL-10, and AMPs gene. Meanwhile increased HMGN2 represses expression of TJs proteins such as ZO-1, Occludin, E-Cadherin and Claudin-1 to disrupt paracellular TJs’ ultrastructure and permeability. Thus, invading bacteria may pass through paracellular routes by up-regulating host HMGN2 expression in urothelium.

Article Snippet: The following antibodies were used: rabbit polyclonal HMGN2 antibody (Cell Signalling Technology), mouse monoclonal GAPDH antibody (Beyotime Biotechnology), Rabbit monoclonal Occludin antibody (invitrogen, Thermofisher scientific), ZO-1 (invitrogen, Thermofisher scientific), E-cadherin (Cell Signalling Technology).

Techniques: Expressing, Permeability, Bacteria