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Cell Signaling Technology Inc rabbit polyclonal antibodies against hmgn2
Primer sequences for RT‐qPCR.

Rabbit Polyclonal Antibodies Against Hmgn2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 93 stars, based on 15 article reviews

rabbit polyclonal antibodies against hmgn2 - by Bioz Stars, 2026-07

93/100 stars

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1) Product Images from "The regulatory effect of acetylation of HMGN2 and H3K27 on pyocyanin‐induced autophagy in macrophages by affecting Ulk1 transcription"

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

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.16788

Figure Legend Snippet: Primer sequences for RT‐qPCR.

Techniques Used:

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

Techniques Used: Mutagenesis

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. <xref ref-type=

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 " title="... of the DMSO group. (C) IP showing the HMGN2 with (D) densitometric analysis showing the relative expression ..." property="contentUrl" width="100%" height="100%"/>
Figure Legend 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

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

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

Figure Legend 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

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

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

Figure Legend 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

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

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

Figure Legend 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

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

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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.

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

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

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

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

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

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)).

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

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)).

Journal: Acta biochimica Polonica

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)).

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

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)).

Journal: Acta biochimica Polonica

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)).

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

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.

Journal: Acta biochimica Polonica

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.

Techniques: Expressing, Permeability, Bacteria

$Validation of potential novel Aurora B phosphorylation sites and substrate. A, in vitro kinase assay with recombinant His-INCENP/GST-tagged Aurora B (wild-type and kinase-dead), in the presence of ATPγS. Recombinant GST-INCENP 1–80, GST-INCENP 1–80S72A, or histone H3 was added as substrate. B, U2OS cells were transfected with either empty GFP vector (ev) or a plasmid encoding GFP-HMGN2. Mitotic cells were collected by mitotic shake-off, and the remaining adherent cells were used as interphase cell input. The overexpressed proteins were immunoprecipitated from the interphase or mitotic cell lysates with an anti-GFP antibody. The precipitated proteins were separated by SDS-PAGE, and Western blots were probed with an antibody specific for phosphorylated Ser-25/Ser-29 in HMGN2 (middle panel) and subsequently reprobed with an anti-GFP antibody (upper panel). Western blots of whole cell extracts were probed with an antibody specific for phosphorylated histone H3 (Ser-10, lower panel). C and D, U2OS cells were released into nocodazole in the presence or absence of the indicated Aurora B inhibitors. HMGN2, Aurora B, and centromeres (CREST) were visualized with specific antibodies. Images of fields of mitotic cells were captured by a Zeiss LSM microscope (C), and the fraction of mitotic cells with no, low, or high levels of HMGN2 on mitotic chromosomes was quantified (D). For each condition >100 cells were counted. Scale bar = 20 micrometer.$

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Development of a Chemical Genetic Approach for Human Aurora B Kinase Identifies Novel Substrates of the Chromosomal Passenger Complex *

doi: 10.1074/mcp.M111.013912

Figure Lengend Snippet: Validation of potential novel Aurora B phosphorylation sites and substrate. A, in vitro kinase assay with recombinant His-INCENP/GST-tagged Aurora B (wild-type and kinase-dead), in the presence of ATPγS. Recombinant GST-INCENP 1–80, GST-INCENP 1–80S72A, or histone H3 was added as substrate. B, U2OS cells were transfected with either empty GFP vector (ev) or a plasmid encoding GFP-HMGN2. Mitotic cells were collected by mitotic shake-off, and the remaining adherent cells were used as interphase cell input. The overexpressed proteins were immunoprecipitated from the interphase or mitotic cell lysates with an anti-GFP antibody. The precipitated proteins were separated by SDS-PAGE, and Western blots were probed with an antibody specific for phosphorylated Ser-25/Ser-29 in HMGN2 (middle panel) and subsequently reprobed with an anti-GFP antibody (upper panel). Western blots of whole cell extracts were probed with an antibody specific for phosphorylated histone H3 (Ser-10, lower panel). C and D, U2OS cells were released into nocodazole in the presence or absence of the indicated Aurora B inhibitors. HMGN2, Aurora B, and centromeres (CREST) were visualized with specific antibodies. Images of fields of mitotic cells were captured by a Zeiss LSM microscope (C), and the fraction of mitotic cells with no, low, or high levels of HMGN2 on mitotic chromosomes was quantified (D). For each condition >100 cells were counted. Scale bar = 20 micrometer.

Article Snippet: For HMGN2 immunofluorescence, a brief PEM/Triton 0.2% pre-extraction was performed prior to fixation with 4% PFA, and rabbit anti-HMGN2 polyclonal antibody (Acris Antibodies) was used as primary antibody.

Techniques: In Vitro, Kinase Assay, Recombinant, Transfection, Plasmid Preparation, Immunoprecipitation, SDS Page, Western Blot, Microscopy

Aurora B substrates unique to this screen The criteria for inclusion include phosphopeptide(s) found in two independent experiments, R X (S/T) motif, E-score ≤ 10E-4, SLIP score ≥ 6, and substrate not found by Kettenbach et al. ( 30 ).

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Development of a Chemical Genetic Approach for Human Aurora B Kinase Identifies Novel Substrates of the Chromosomal Passenger Complex *

doi: 10.1074/mcp.M111.013912

Figure Lengend Snippet: Aurora B substrates unique to this screen The criteria for inclusion include phosphopeptide(s) found in two independent experiments, R X (S/T) motif, E-score ≤ 10E-4, SLIP score ≥ 6, and substrate not found by Kettenbach et al. ( 30 ).

Techniques: Binding Assay

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