rabbit anti hp1γ  (Novus Biologicals)

Journal: Life science alliance

Article Title: Oral bacteria induce IgA autoantibodies against a mesangial protein in IgA nephropathy model mice.

doi: 10.26508/lsa.202402588

Figure Lengend Snippet: Figure 1. Identification of MC antigens reactive to IgA auto-Abs generated in gddY mice. (A) Detection of antigens for rmAbs derived from gddY mouse PBs in the lysates of gddY mouse primary MCs by WB. Split membranes, each containing proteins in one lane, were incubated with the indicated rmAbs (1 μg/ml), followed by an anti-human IgG Ab. (B) Immunoprecipitation of gddY mouse MC lysates with rmAb#66 or the control rmAb#NP. SDS–PAGE of the precipitates was developed using silver staining. The bands indicated by arrows were excised and analyzed using mass spectrometry. Those from the rmAb#66 precipitate included CBX1, CBX3, and CBX5, as indicated by arrows. (C) FLAG-tagged CBX1, CBX3, or CBX5 proteins transiently expressed in HEK293T cells were detected by WB with the indicated rmAbs (1 μg/ml), followed by anti-human IgG Ab (left panel) or anti-FLAG Ab (right panel). (D) ELISA screening of rmAbs for binding to CBX proteins. The heatmap represents the resulting OD values of the indicated rmAbs, with “+” indicating a saturated signal. (E) Reactivity of gddY mouse serum IgA with the CBX proteins. WB was performed as in (C) using sera of individual BALB/c or gddY mice as the primary Ab as indicated and anti-IgA Ab as the secondary Ab (left, top) or anti-FLAG Ab to confirm the presence of CBX proteins on the blot (left, bottom). Representative data are shown here. Venn diagram showing the percentage of gddY mice whose serum IgA reacted with CBX1, CBX3, or CBX5 (right panel) (n = 14). (F) ELISA determination of anti-CBX3 IgA Abs in sera from healthy controls (HC; n = 30) and patients with IgAN (IgAN; n = 70). ***: 0.0006 (Mann–Whitney U test). The dashed line shows 99% confidence interval (CI) of the control sera. One of two or three independent experiments with similar results is shown in each panel. Source data are available for this figure.

Article Snippet: For FCM, the following Abs were used: anti-mouse IgA-APC (clone 11- 44-2; Southern Biotech), anti-mouse CD73-biotin (clone TY/11.8; BioLegend), anti-mouse CD45-PerCP-Cy5.5 (clone 30-F11; BioLegend), anti-mouse CD31-PE (clone 390; BioLegend), anti-mouse TER119BV510 (clone TER-119; BioLegend), rabbit polyclonal anti-HP1γ (CBX3) Ab (2619; Cell Signaling Technology), rabbit monoclonal anti-HP1β (CBX1) Ab (8676; Cell Signaling Technology), rabbit IgG (for isotypematched control; 011-000-003; Jackson ImmunoResearch), antirabbit IgG-BV421 (clone Poly4064; BioLegend), and anti-human IgG Fcγ-Alexa Fluor 647 (709-606-098; Jackson ImmunoResearch).

Techniques: Generated, Derivative Assay, Incubation, Immunoprecipitation, Control, SDS Page, Silver Staining, Mass Spectrometry, Enzyme-linked Immunosorbent Assay, Binding Assay, MANN-WHITNEY

Journal: Life science alliance

Article Title: Oral bacteria induce IgA autoantibodies against a mesangial protein in IgA nephropathy model mice.

doi: 10.26508/lsa.202402588

Figure Lengend Snippet: Figure 2. CBX3 is an autoantigen expressed on the surfaces of MCs. (A, B) Representative histograms of FCM analysis of glomerular CD45– cells from BALB/c mice stained with Abs against CD45, CD73, and CD31 for gating on MCs (CD73+

Article Snippet: For FCM, the following Abs were used: anti-mouse IgA-APC (clone 11- 44-2; Southern Biotech), anti-mouse CD73-biotin (clone TY/11.8; BioLegend), anti-mouse CD45-PerCP-Cy5.5 (clone 30-F11; BioLegend), anti-mouse CD31-PE (clone 390; BioLegend), anti-mouse TER119BV510 (clone TER-119; BioLegend), rabbit polyclonal anti-HP1γ (CBX3) Ab (2619; Cell Signaling Technology), rabbit monoclonal anti-HP1β (CBX1) Ab (8676; Cell Signaling Technology), rabbit IgG (for isotypematched control; 011-000-003; Jackson ImmunoResearch), antirabbit IgG-BV421 (clone Poly4064; BioLegend), and anti-human IgG Fcγ-Alexa Fluor 647 (709-606-098; Jackson ImmunoResearch).

Techniques: Staining

Journal: Life science alliance

Article Title: Oral bacteria induce IgA autoantibodies against a mesangial protein in IgA nephropathy model mice.

doi: 10.26508/lsa.202402588

Figure Lengend Snippet: Figure 5. The gddY auto-Abs reacted with commensal bacteria in the oral cavity of the gddY mice. (A, B) WB analysis of lysates of bacteria from feces (fecal), small intestine (SI), and oral cavity (oral) of gddY mice blotted with sera from gddY mice (A) or culture supernatant of leukocytes from gddY mouse kidneys (B) as the primary Abs and anti-IgA as the secondary Ab. The IgA concentration in the primary Abs was adjusted to 1 μg/ml by dilution. The bacteria were cultured anaerobically on brain heart infusion agar before lysis. (C, D, E, F) FCM analyses of cultured bacteria stained with various Ab solutions (containing 1 μg/ml IgA) and SYBR Green (for bacterial DNA). (C, D) Bacteria from the oral cavity (oral) or small intestine (C), or oral bacteria from BALB/c or gddY mice (D), were stained with sera from gddY mice. (E) Oral bacteria from gddY mice were stained with the culture supernatant of leukocytes from the indicated organs of gddY mice. (C, D, E) APC-conjugated anti-mouse IgA was used as secondary Ab. The secondary Ab alone did not stain cultured bacteria (data not shown). (F) Oral or fecal bacteria from gddY mice cultured under the indicated conditions were stained with rmAb#66 or rmAb#NP, followed by Alexa Fluor 647–anti-human IgG. (C, D, E) The frequencies of IgA-bound bacteria among whole bacteria stained with SYBR Green are plotted. Data are presented as means ± SD of biological replicates. (G) rmAb#66 (1 μg/ml) was incubated with the indicated concentrations of recombinant CBX3 or OVA in equal volumes for 20 min at RT, mixed with cultured oral bacteria from gddY mice, and incubated overnight on ice. The bacteria were stained with Alexa Fluor 647-anti-human IgG to detect bound rmAb#66, together with SYBR Green to label the bacteria, and analyzed by FCM. The frequencies of rmAb#66-bound bacteria among all bacteria were plotted. Data are presented as means ± SD of three technical replicates. *P < 0.05; ***P < 0.005; ****P < 0.001. P-values were calculated using unpaired t test. One of two independent experiments with similar results is shown in each panel. Source data are available for this figure.

Article Snippet: For FCM, the following Abs were used: anti-mouse IgA-APC (clone 11- 44-2; Southern Biotech), anti-mouse CD73-biotin (clone TY/11.8; BioLegend), anti-mouse CD45-PerCP-Cy5.5 (clone 30-F11; BioLegend), anti-mouse CD31-PE (clone 390; BioLegend), anti-mouse TER119BV510 (clone TER-119; BioLegend), rabbit polyclonal anti-HP1γ (CBX3) Ab (2619; Cell Signaling Technology), rabbit monoclonal anti-HP1β (CBX1) Ab (8676; Cell Signaling Technology), rabbit IgG (for isotypematched control; 011-000-003; Jackson ImmunoResearch), antirabbit IgG-BV421 (clone Poly4064; BioLegend), and anti-human IgG Fcγ-Alexa Fluor 647 (709-606-098; Jackson ImmunoResearch).

Techniques: Bacteria, Concentration Assay, Cell Culture, Lysis, Staining, SYBR Green Assay, Incubation, Recombinant

Journal: Life science alliance

Article Title: Oral bacteria induce IgA autoantibodies against a mesangial protein in IgA nephropathy model mice.

doi: 10.26508/lsa.202402588

Figure Lengend Snippet: Figure 6. Identification of a bacterial strain that shares an rmAb#66 epitope with CBX3. (A) Isolated bacteria (C42) were stained with rmAb#66 or rmAb#NP (1 μg/ml), followed by Alexa Fluor 647–anti-human IgG together with SYBR Green, and analyzed by FCM. (B) rmAb#66 (1 μg/ml) was incubated with CBX3 or OVA and then with C42, which was stained with anti-human IgG and analyzed as described in Fig 5G. Data are presented as means ± SD of two technical replicates. (C) C42 bacteria (2 × 106 cells) were incubated with the indicated concentrations of PNGase F (Roche) for 4 h at 37°C, stained with rmAb#66 (1 μg/ml) overnight on ice, and labeled with SYBR Green. Binding of rmAb#66 to C42 was detected by FCM using Alexa Fluor 647-anti-human IgG (n = 2). (D) BALB/c mice were immunized s.c. with UV-killed C42 or E. faecalis, together with zymosan as an adjuvant, at 4 wk of age. WB analysis was performed as shown in Fig 3E (except that CBX1 and CBX5 samples were omitted) with sera from immunized mice at 8 wk of age as the primary Abs and anti-IgG Ab as the secondary Ab (left panel) or anti-FLAG Ab (right panel). (E) Representative IFM images of kidney sections from the mice used in (D). Kidney sections were stained with an anti-IgA Ab (red). Dashed circles indicate areas of glomeruli and white lines indicate scale bars (100 μm). (F) Frequency of bacteria recognized by rmAb#66 among all bacteria (SYBR Green+) in the oral cavity of gddY mice (n = 6). Bacteria from gddY mice fed a normal diet (control) or an ED (ED) were cultured anaerobically on brain heart infusion agar, stained with rmAb#66 (1 μg/ml) and Alexa Fluor 647–anti-human IgG, together with SYBR Green, and analyzed by FCM. (G) Frequency of each bacterial strain among the total bacteria in the oral cavities of gddY mice fed a normal diet (control) or an ED, as determined by 16S rRNA bulk sequencing (n = 6). An underscore after each genus name indicates that the species was not classified. Data are presented as means ± SD of biological replicates. *P < 0.05; ***P < 0.005; ****P < 0.001. P-values were calculated using the unpaired t test. One of two independent experiments with similar results is shown in each panel. Source data are available for this figure.

Article Snippet: For FCM, the following Abs were used: anti-mouse IgA-APC (clone 11- 44-2; Southern Biotech), anti-mouse CD73-biotin (clone TY/11.8; BioLegend), anti-mouse CD45-PerCP-Cy5.5 (clone 30-F11; BioLegend), anti-mouse CD31-PE (clone 390; BioLegend), anti-mouse TER119BV510 (clone TER-119; BioLegend), rabbit polyclonal anti-HP1γ (CBX3) Ab (2619; Cell Signaling Technology), rabbit monoclonal anti-HP1β (CBX1) Ab (8676; Cell Signaling Technology), rabbit IgG (for isotypematched control; 011-000-003; Jackson ImmunoResearch), antirabbit IgG-BV421 (clone Poly4064; BioLegend), and anti-human IgG Fcγ-Alexa Fluor 647 (709-606-098; Jackson ImmunoResearch).

Techniques: Isolation, Bacteria, Staining, SYBR Green Assay, Incubation, Labeling, Binding Assay, Adjuvant, Control, Cell Culture, Sequencing

Journal: Life science alliance

Article Title: Oral bacteria induce IgA autoantibodies against a mesangial protein in IgA nephropathy model mice.

doi: 10.26508/lsa.202402588

Figure Lengend Snippet: Figure 7. Determination of rmAb#66 epitope on CBX3. (A) Schematic representation of CBX3 proteins: 1/2 CBX3 (AA1-92) and 2/3 CBX3 (AA63-184). (B, C) FLAG-tagged 1/2 or 2/3 CBX3 proteins, transiently expressed in HEK293T cells, were detected by WB with rmAb#66 or rmAb#NP (1 μg/ml) followed by anti-human IgG Ab ((B), left panel). The same membrane was reblotted with sera of two gddY mice followed by anti-mouse IgA Ab ((C), left panel). Protein loading was confirmed using an anti-FLAG Ab ((B, C), right panels; the same blot is shown). (D) Amino acid sequence alignment of CBX proteins (AA1-62) (top). WB analysis of FLAG-tagged WT or the indicated mutated CBX3 proteins transiently expressed in HEK293T cells, performed as in (B) (bottom). (E) Reactivity to CBX3 (WT) or CBX3 mutant 54FA (54FA) proteins of the indicated rmAbs at the indicated concentrations was evaluated by ELISA and presented as OD450 values. One of two independent experiments with similar results is shown in each panel. Source data are available for this figure.

Article Snippet: For FCM, the following Abs were used: anti-mouse IgA-APC (clone 11- 44-2; Southern Biotech), anti-mouse CD73-biotin (clone TY/11.8; BioLegend), anti-mouse CD45-PerCP-Cy5.5 (clone 30-F11; BioLegend), anti-mouse CD31-PE (clone 390; BioLegend), anti-mouse TER119BV510 (clone TER-119; BioLegend), rabbit polyclonal anti-HP1γ (CBX3) Ab (2619; Cell Signaling Technology), rabbit monoclonal anti-HP1β (CBX1) Ab (8676; Cell Signaling Technology), rabbit IgG (for isotypematched control; 011-000-003; Jackson ImmunoResearch), antirabbit IgG-BV421 (clone Poly4064; BioLegend), and anti-human IgG Fcγ-Alexa Fluor 647 (709-606-098; Jackson ImmunoResearch).

Techniques: Membrane, Sequencing, Mutagenesis, Enzyme-linked Immunosorbent Assay