Journal: Journal of Lipid Research

Article Title: Interleukin-4 regulates lipid metabolism by inhibiting adipogenesis and promoting lipolysis

doi: 10.1194/jlr.M041392

Figure Lengend Snippet: Effects of IL-4 on adipogenesis of 3T3-L1 cells. Lipid accumulation and expression levels of adipocyte-specific genes during adipogenesis under IL-4 treatment were analyzed. A: 3T3-L1 cells were induced into mature adipocytes in the absence or presence of 10 ng/ml of IL-4 and subjected to ORO staining. B: Quantification of the ORO staining results by measuring the corresponding absorbance of isopropanol-extracted dye at 490 nm. C: Protein expression of aP2, C/EBPα, C/EBPβ, and PPARγ in 3T3-L1 was analyzed at the indicated time. β-actin served as a loading control. D: Quantification of Western blotting by densitometer. The data are presented as the mean ± SEM (n = 7). * P < 0.05 and ** P < 0.01 compared with MDI. E, F: mRNA expression of the aP2, C/EBPα ( Cebpa ), PPARγ ( Pparg ), GLUT4 ( Glut4 ), and adipocyte-specific genes (adiponectin, Adipoq ; resistin, Retn ; fatty acid synthase, Fasn ; lipoprotein lipase, Lpl ) in 3T3-L1 was analyzed by real-time PCR (E) and RT-PCR (F), respectively, at the indicated time. All results were normalized to 18s rRNA. The data are presented as the mean ± SEM (n = 3), and statistically analyzed by two-tailed unpaired Student's t -test. * P < 0.05 compared with MDI. CON, control; D, day.

Article Snippet: The results showed that while lipid accumulation was significantly suppressed by exposing the cells to IL-4 during the entire differentiation process (MDI/IL-4), short-term IL-4 exposure at the MCE stage (MDI/IL-4-MCE) was sufficient to inhibit adipogenesis ( ).

Techniques: Expressing, Staining, Control, Western Blot, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

Journal: Journal of Lipid Research

Article Title: Interleukin-4 regulates lipid metabolism by inhibiting adipogenesis and promoting lipolysis

doi: 10.1194/jlr.M041392

Figure Lengend Snippet: IL-4 inhibits adipogenesis at early MCE stage. Effect of IL-4 inhibitory effect on differentiation under various schemes of IL-4 treatment was analyzed. A, B: PPARγ agonist TZD rescued adipogenesis under IL-4 treatment. 3T3-L1 cells were treated with 10 μM TZD on day 4, and lipid accumulation was examined by ORO staining on day 8. B: The ORO staining in cells was extracted with isopropanol and quantitated at optical density 490 nm. * P < 0.05 DMSO versus TZD; ** P < 0.01 MDI versus MDI/IL-4. Data are presented as the mean ± SEM (n = 3). C, D: Schematic illustrator indicates the different IL-4 exposure periods of adipocytes during differentiation (PRE, IL-4 treatment from day −2 to 0; MCE, IL-4 treatment from day 0 to 2; POST, IL-4 treatment from day 2 to 8). C: Intracellular lipid contents were stained by ORO on day 8. Scale bar, 10 μm. D: The corresponding lipid contents of staining results were measured by spectrophotometer. Data are presented as the mean ± SEM (n = 3). *** P < 0.001 compared with MDI. E: Expression of C/EBPγ and PPARγ in adipocytes with differential IL-4 treatment as indicated in (C) was analyzed. Briefly, cells were treated with IL-4 as indicated, and then cell lysates were harvested on day 8 and subjected to Western blotting with β-actin as loading control.

Article Snippet: The results showed that while lipid accumulation was significantly suppressed by exposing the cells to IL-4 during the entire differentiation process (MDI/IL-4), short-term IL-4 exposure at the MCE stage (MDI/IL-4-MCE) was sufficient to inhibit adipogenesis ( ).

Techniques: Staining, Spectrophotometry, Expressing, Western Blot, Control

Journal: Journal of Lipid Research

Article Title: Interleukin-4 regulates lipid metabolism by inhibiting adipogenesis and promoting lipolysis

doi: 10.1194/jlr.M041392

Figure Lengend Snippet: IL-4 partially inhibits cell cycle progression at the MCE stage. Expression levels of transcription factors and cell proliferation activity during the MCE stage under IL-4 treatment were analyzed. A: mRNA expression of C/EBPβ ( Cebpb ) and C/EBPδ ( Cebpd ) in 3T3-L1 cells was analyzed by real-time PCR at the indicated time in the absence or presence of 10 ng/ml IL-4. B: 3T3-L1 cells were induced into differentiation in the absence or presence of IL-4, and then cells were trypsinized, stained with trypan blue, and counted at the indicated time. Data are presented as the mean ± SEM (n = 9). * P < 0.05 MDI/IL-4 versus MDI. C: Expression of p27 Kip was analyzed at the indicated time in the absence or presence of IL-4 treatment by Western blotting with β-actin as loading control. Quantitative results are presented as the mean ± SEM (n = 3). * P < 0.05 compared with MDI.

Article Snippet: The results showed that while lipid accumulation was significantly suppressed by exposing the cells to IL-4 during the entire differentiation process (MDI/IL-4), short-term IL-4 exposure at the MCE stage (MDI/IL-4-MCE) was sufficient to inhibit adipogenesis ( ).

Techniques: Expressing, Activity Assay, Real-time Polymerase Chain Reaction, Staining, Western Blot, Control

Journal: Journal of Lipid Research

Article Title: Interleukin-4 regulates lipid metabolism by inhibiting adipogenesis and promoting lipolysis

doi: 10.1194/jlr.M041392

Figure Lengend Snippet: IL-4 induces STAT6 signaling pathway during adipogenesis. Expression and phosphorylation levels of molecules in the signaling pathway at the MCE stage under IL-4 treatment were analyzed. A: 3T3-L1 cells were induced to differentiate with MDI cocktail in the absence or presence of 10 ng/ml IL-4. Cell lysates were harvested at the indicated times and then subjected to Western blotting analysis using antibodies against STAT6, phospho-STAT6 (Tyr 641 ), phospho-ERK1/2, total ERK1/2, pGSK3β, and total GSK3β. B: Cells were allowed to differentiate in the absence or presence of 10 ng/ml IL-4. The nuclear extracts were harvested at the indicated times and then subjected to EMSA experiments. The upper arrow indicates the STAT6-probe complex. The lower arrow indicates the unbound free probe. C: Pretreatment with AG490 (10 μM) rescued IL-4-inhibited adipogenesis. 3T3-L1 cells were pretreated with 10 μM AG490 and then induced to differentiate in the absence or presence of IL-4. Lipid accumulation was observed by ORO staining and quantified. Scale bar, 20 μm. *** P < 0.001. D: 3T3-L1 cells were transfected with 100 nM STAT6 siRNA for 24 h and then subjected to differentiation in the absence or presence of 10 ng/ml IL-4. Lipid accumulation was observed by ORO staining on day 8 and quantified. Scale bar, 10 μm. *** P < 0.001. E: Expression of PPARγ and C/EBPα in 3T3-L1 cells transfected with 100 nM nonspecific siRNA or STAT6 siRNA. Data are presented as the mean ± SEM (n = 9). CON, control.

Article Snippet: The results showed that while lipid accumulation was significantly suppressed by exposing the cells to IL-4 during the entire differentiation process (MDI/IL-4), short-term IL-4 exposure at the MCE stage (MDI/IL-4-MCE) was sufficient to inhibit adipogenesis ( ).

Techniques: Expressing, Phospho-proteomics, Western Blot, Staining, Transfection, Control

Journal: Journal of Lipid Research

Article Title: Interleukin-4 regulates lipid metabolism by inhibiting adipogenesis and promoting lipolysis

doi: 10.1194/jlr.M041392

Figure Lengend Snippet: IL-4 enhances basal and ISO-stimulated lipolysis in mature adipocytes. Effects of IL-4 on lipolysis and HSL expression/activity in 3T3-L1 mature adipocytes were analyzed. A: Left panel: Cells were treated with 10 ng/ml IL-4 for 24 h. Culture medium was collected and glycerol content was determined. Data are presented as the mean ± SEM (n = 8). ** P < 0.01 compared with untreated control. Right panel: HSL, ATGL, and perilipin protein levels in cells after 24 h of 10 ng/ml IL-4 treatment were analyzed by Western blotting with β-actin as a loading control. B, C: Cells were treated with 10 ng/ml IL-4 for 3 h followed by 10 μM ISO for 1 h. Culture medium was collected and glycerol and NEFA contents were determined. Data are presented as the mean ± SEM (n = 8). ** P < 0.01. D: Upper panel: cells were treated with IL-4 in the absence or presence of ISO. Cell lysates were harvested and subjected to Western blotting analysis using antibodies against phospho-PKA substrate, Ser 563 -pHSL, and total HSL with β-actin as a loading control. Lower panel: quantitative results are presented as the mean ± SEM (n = 3). ** P < 0.01 and *** P < 0.001. E: Ser 563 -pHSL was recruited to the LD surface (arrows). Cells were treated with vehicle (DMSO), 10 ng/ml IL-4, 10 μM ISO, or 10 μM ISO with 10 ng/ml IL-4 for 1 h prior to fixation. Cells were immunolabeled with Ser 563 -pHSL (red), LDs (BODIPY, green), and DAPI (blue). Scale bar, 20 μm. Mean fluorescence intensity (MFI) of the cells and LDs were analyzed using ZEN 2009 software. Data are presented as mean ± SEM of three separate experiments. * P < 0.05 and ** P < 0.01.

Article Snippet: The results showed that while lipid accumulation was significantly suppressed by exposing the cells to IL-4 during the entire differentiation process (MDI/IL-4), short-term IL-4 exposure at the MCE stage (MDI/IL-4-MCE) was sufficient to inhibit adipogenesis ( ).

Techniques: Expressing, Activity Assay, Control, Western Blot, Immunolabeling, Fluorescence, Software

Journal: Journal of Lipid Research

Article Title: Interleukin-4 regulates lipid metabolism by inhibiting adipogenesis and promoting lipolysis

doi: 10.1194/jlr.M041392

Figure Lengend Snippet: IL-4 promotes perilipin phophorylation in mature adipocytes via the PKA pathway. Expression and phosphorylation levels of perilipin in 3T3-L1 mature adipocytes under IL-4 treatment were analyzed. A: Cells were treated either with 10 ng/ml IL-4 or combined with 10 μM ISO treatment for 1 h. Cell lysates were harvested and subjected to Western blotting analysis using antibodies against phospho-PKA substrate and perilipin with β-actin as a loading control. Data are presented as mean ± SEM of three separate experiments. * P < 0.05. B, C: The 62 kDa phospho-PKA substrate was identified as perilipin. B: Cells were treated with 10 ng/ml IL-4 for 1 h. Perilipin in the cell extracts was first depleted by anti-perilipin antibodies, and then the perilipin-cleared supernatant was harvested and subjected to Western blot analysis. C: Cell lysates after IL-4 treatment were immunoprecipitated with anti-phosho-PKA substrate antibody and subjected to Western blot analysis. D: Cells were pretreated with 20 μM PKA inhibitor H-89 for 1 h and followed by IL-4 treatment in the absence or presence of 10 μM ISO for 1 h. Cell lysates were harvested and subjected to Western blotting analysis using antibodies against phospho-PKA substrate with β-actin as a loading control.

Article Snippet: The results showed that while lipid accumulation was significantly suppressed by exposing the cells to IL-4 during the entire differentiation process (MDI/IL-4), short-term IL-4 exposure at the MCE stage (MDI/IL-4-MCE) was sufficient to inhibit adipogenesis ( ).

Techniques: Expressing, Phospho-proteomics, Western Blot, Control, Immunoprecipitation

Journal: Scientific Reports

Article Title: A complex of novel protease inhibitor, ovostatin homolog, with its cognate proteases in immature mice uterine luminal fluid

doi: 10.1038/s41598-019-41426-4

Figure Lengend Snippet: Purification of the ULF gelatinolytic enzyme. ( A ) Gelatin zymogram of immature mouse ULF. The ULF (6 μl) was analyzed by gelatin zymography. ( B ) Elution profile of ULF gelatinolytic enzyme carried out by gel filtration chromatography. Soluble ULF (1 ml) was subjected to gel filtration chromatography on a Sephacryl S-400 column. The elution was monitored by UV spectrophotometry with absorbance at 280 nm (—). The gelatinolytic activity of 26 kDa enzyme in each indicated fraction was quantitatively detected (---). Arrows indicate the protein markers eluted from the same column. The protein markers are human α 2 MG (725 kDa), apoferritin (443 kDa) and carbonic anhydrase (29 kDa). The horizontal bar represents the pooled fractions for further purification by the DEAE-Sepharose chromatography. ( C ) Elution profile of DEAE-Sepharose chromatography. The pooled fractions are marked by a horizontal bar. ( D ) Gelatin zymographic assay. Lane 1: immature mouse ULF (6 μl). Lane 2: the pooled fractions (0.3 ml) from gel filtration. Line 3: the pooled fractions (0.25 ml) from DEAE-Sepharose chromatography.

Article Snippet: Sephacryl S-400, DEAE-Sepharose, glutathione Sepharose 4B, CNBr-activated Sepharose 4B, polyvinylidene difluoride (PVDF) membrane, and pGEX-4T-1 were ordered from GE Healthcare Life Sciences (Piscataway, NJ, USA).

Techniques: Purification, Zymography, Filtration, Chromatography, Spectrophotometry, Activity Assay

Journal: Scientific Reports

Article Title: A complex of novel protease inhibitor, ovostatin homolog, with its cognate proteases in immature mice uterine luminal fluid

doi: 10.1038/s41598-019-41426-4

Figure Lengend Snippet: mOH is present in immature mouse ULF. ( A ) The immature mouse ULF and the gelatinolytic enzyme complex were separated by non-reducing SDS-PAGE and then analyzed by Western blotting, probed for mOH by POHA. The POHA was purified from rabbit anti-mOH sera. Lane 1: immature mouse ULF (5 μl). Lane 2: the gelatinolytic enzyme complex (0.5 μg) that was the resulting product after immature mouse ULF was purified by gel filtration and DEAE-Sepharose chromatography. ( B ) The immature mouse ULF and the gelatinolytic enzyme complex were separated by reducing SDS-PAGE and then analyzed by Western blotting, probed for reducing mOH by rabbit anti-GST-mOH[870–940] sera. Lane 1: immature mouse ULF (5 μl). Lane 2: the gelatinolytic enzyme complex (0.5 μg).

Article Snippet: Sephacryl S-400, DEAE-Sepharose, glutathione Sepharose 4B, CNBr-activated Sepharose 4B, polyvinylidene difluoride (PVDF) membrane, and pGEX-4T-1 were ordered from GE Healthcare Life Sciences (Piscataway, NJ, USA).

Techniques: SDS Page, Western Blot, Purification, Filtration, Chromatography

Journal: Scientific Reports

Article Title: A complex of novel protease inhibitor, ovostatin homolog, with its cognate proteases in immature mice uterine luminal fluid

doi: 10.1038/s41598-019-41426-4

Figure Lengend Snippet: A complex of mOH with three gelatinolytic enzymes in immature mouse ULF. ( A ) Co-elution of mOH with gelatinolytic enzymes during purification of mOH by gel filtration. Each indicated fraction (0.3 ml) was separated by SDS-PAGE and then applied to immunoblot analysis for detection of mOH protein using POHA (upper panel) and for detection of mCLCA3 using anti-mCLCA3 antibody (middle panel), respectively. The identical samples were also subjected to gelatin zymography for detection of gelatinolytic enzymes (lower panel). The original immunoblots and scan of gelatin zymogram were displayed in Supplementary Fig. . ( B ) Co-immunoprecipitation of mOH with three gelatinolytic enzymes. Either POHA or preimmune antibody (PIA) was pre-immobilized on Protein A-Sepharose (60 μl), followed by incubation with immature mice ULF (30 μl). After centrifugation, the pellet was re-suspended in 30 μl of sample buffer. An aliquot of re-suspended pellet or supernatant was subjected to SDS-PAGE, followed by gelatin zymography for detection of gelatinolytic enzymes. Lane 1: PIA-treated ULF supernatant (6 μl). Lane 2: PIA-treated ULF pellet (6 μl). Lane 3: POHA-treated ULF supernatant (6 μl), Lane 4: POHA-treated ULF pellet (6 μl).

Article Snippet: Sephacryl S-400, DEAE-Sepharose, glutathione Sepharose 4B, CNBr-activated Sepharose 4B, polyvinylidene difluoride (PVDF) membrane, and pGEX-4T-1 were ordered from GE Healthcare Life Sciences (Piscataway, NJ, USA).

Techniques: Co-Elution Assay, Purification, Filtration, SDS Page, Western Blot, Zymography, Immunoprecipitation, Incubation, Centrifugation

Journal: Scientific Reports

Article Title: A complex of novel protease inhibitor, ovostatin homolog, with its cognate proteases in immature mice uterine luminal fluid

doi: 10.1038/s41598-019-41426-4

Figure Lengend Snippet: Tissue distribution of mOH mRNA. Total RNAs isolated from various adult mice tissues or from different stages of mice embryos were subjected to RT-PCR. The resulting products were separated in agarose gel and visualized by ethidium bromide staining under exposure of UV light. The RT-PCR product of GAPDH was served as the internal control. The original agarose gels were shown in Supplementary Fig. .

Article Snippet: Sephacryl S-400, DEAE-Sepharose, glutathione Sepharose 4B, CNBr-activated Sepharose 4B, polyvinylidene difluoride (PVDF) membrane, and pGEX-4T-1 were ordered from GE Healthcare Life Sciences (Piscataway, NJ, USA).

Techniques: Isolation, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining

Journal: Scientific Reports

Article Title: A complex of novel protease inhibitor, ovostatin homolog, with its cognate proteases in immature mice uterine luminal fluid

doi: 10.1038/s41598-019-41426-4

Figure Lengend Snippet: Purification of the ULF gelatinolytic enzyme. ( A ) Gelatin zymogram of immature mouse ULF. The ULF (6 μl) was analyzed by gelatin zymography. ( B ) Elution profile of ULF gelatinolytic enzyme carried out by gel filtration chromatography. Soluble ULF (1 ml) was subjected to gel filtration chromatography on a Sephacryl S-400 column. The elution was monitored by UV spectrophotometry with absorbance at 280 nm (—). The gelatinolytic activity of 26 kDa enzyme in each indicated fraction was quantitatively detected (---). Arrows indicate the protein markers eluted from the same column. The protein markers are human α 2 MG (725 kDa), apoferritin (443 kDa) and carbonic anhydrase (29 kDa). The horizontal bar represents the pooled fractions for further purification by the DEAE-Sepharose chromatography. ( C ) Elution profile of DEAE-Sepharose chromatography. The pooled fractions are marked by a horizontal bar. ( D ) Gelatin zymographic assay. Lane 1: immature mouse ULF (6 μl). Lane 2: the pooled fractions (0.3 ml) from gel filtration. Line 3: the pooled fractions (0.25 ml) from DEAE-Sepharose chromatography.

Article Snippet: Sephacryl S-400, DEAE-Sepharose, glutathione Sepharose 4B, CNBr-activated Sepharose 4B, polyvinylidene difluoride (PVDF) membrane, and pGEX-4T-1 were ordered from GE Healthcare Life Sciences (Piscataway, NJ, USA).

Techniques: Purification, Zymography, Filtration, Chromatography, Spectrophotometry, Activity Assay

Journal: Scientific Reports

Article Title: A complex of novel protease inhibitor, ovostatin homolog, with its cognate proteases in immature mice uterine luminal fluid

doi: 10.1038/s41598-019-41426-4

Figure Lengend Snippet: mOH is present in immature mouse ULF. ( A ) The immature mouse ULF and the gelatinolytic enzyme complex were separated by non-reducing SDS-PAGE and then analyzed by Western blotting, probed for mOH by POHA. The POHA was purified from rabbit anti-mOH sera. Lane 1: immature mouse ULF (5 μl). Lane 2: the gelatinolytic enzyme complex (0.5 μg) that was the resulting product after immature mouse ULF was purified by gel filtration and DEAE-Sepharose chromatography. ( B ) The immature mouse ULF and the gelatinolytic enzyme complex were separated by reducing SDS-PAGE and then analyzed by Western blotting, probed for reducing mOH by rabbit anti-GST-mOH[870–940] sera. Lane 1: immature mouse ULF (5 μl). Lane 2: the gelatinolytic enzyme complex (0.5 μg).

Article Snippet: Sephacryl S-400, DEAE-Sepharose, glutathione Sepharose 4B, CNBr-activated Sepharose 4B, polyvinylidene difluoride (PVDF) membrane, and pGEX-4T-1 were ordered from GE Healthcare Life Sciences (Piscataway, NJ, USA).

Techniques: SDS Page, Western Blot, Purification, Filtration, Chromatography

Journal: Scientific Reports

Article Title: A complex of novel protease inhibitor, ovostatin homolog, with its cognate proteases in immature mice uterine luminal fluid

doi: 10.1038/s41598-019-41426-4

Figure Lengend Snippet: A complex of mOH with three gelatinolytic enzymes in immature mouse ULF. ( A ) Co-elution of mOH with gelatinolytic enzymes during purification of mOH by gel filtration. Each indicated fraction (0.3 ml) was separated by SDS-PAGE and then applied to immunoblot analysis for detection of mOH protein using POHA (upper panel) and for detection of mCLCA3 using anti-mCLCA3 antibody (middle panel), respectively. The identical samples were also subjected to gelatin zymography for detection of gelatinolytic enzymes (lower panel). The original immunoblots and scan of gelatin zymogram were displayed in Supplementary Fig. . ( B ) Co-immunoprecipitation of mOH with three gelatinolytic enzymes. Either POHA or preimmune antibody (PIA) was pre-immobilized on Protein A-Sepharose (60 μl), followed by incubation with immature mice ULF (30 μl). After centrifugation, the pellet was re-suspended in 30 μl of sample buffer. An aliquot of re-suspended pellet or supernatant was subjected to SDS-PAGE, followed by gelatin zymography for detection of gelatinolytic enzymes. Lane 1: PIA-treated ULF supernatant (6 μl). Lane 2: PIA-treated ULF pellet (6 μl). Lane 3: POHA-treated ULF supernatant (6 μl), Lane 4: POHA-treated ULF pellet (6 μl).

Article Snippet: Sephacryl S-400, DEAE-Sepharose, glutathione Sepharose 4B, CNBr-activated Sepharose 4B, polyvinylidene difluoride (PVDF) membrane, and pGEX-4T-1 were ordered from GE Healthcare Life Sciences (Piscataway, NJ, USA).

Techniques: Co-Elution Assay, Purification, Filtration, SDS Page, Western Blot, Zymography, Immunoprecipitation, Incubation, Centrifugation

Journal: Scientific Reports

Article Title: A complex of novel protease inhibitor, ovostatin homolog, with its cognate proteases in immature mice uterine luminal fluid

doi: 10.1038/s41598-019-41426-4

Figure Lengend Snippet: Tissue distribution of mOH mRNA. Total RNAs isolated from various adult mice tissues or from different stages of mice embryos were subjected to RT-PCR. The resulting products were separated in agarose gel and visualized by ethidium bromide staining under exposure of UV light. The RT-PCR product of GAPDH was served as the internal control. The original agarose gels were shown in Supplementary Fig. .

Article Snippet: Sephacryl S-400, DEAE-Sepharose, glutathione Sepharose 4B, CNBr-activated Sepharose 4B, polyvinylidene difluoride (PVDF) membrane, and pGEX-4T-1 were ordered from GE Healthcare Life Sciences (Piscataway, NJ, USA).

Techniques: Isolation, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining

Journal: The Journal of Biological Chemistry

Article Title: Autoreactive B cells against malondialdehyde-induced protein cross-links are present in the joint, lung, and bone marrow of rheumatoid arthritis patients

doi: 10.1016/j.jbc.2023.105320

Figure Lengend Snippet: Purified MDA/MAA polyclonal antibodies from RA patients and healthy controls. A , schematic overview of the sequential affinity purification procedure using streptavidin sepharose coupled with biotinylated modified fibrinogen (either MDA-fibrinogen or MDA-SCHB-fibrinogen) for purification of IgG anti-MDA/MAA polyclonal autoantibodies (pAb) from IgG from RA patients or healthy controls. IgG fractions were buffer exchanged to PBS between purification steps and IgG concentrations were assessed with spectrophotometer measurement at 280 nm and ELISA. B , ELISA reactivity of purified anti-MDA/MAA-fibrinogen polyclonal IgG antibodies to MDA-modified HSA (containing both MDA and MAA adducts). C , reactivity of purified IgG anti-MDA-fibrinogen or alternatively anti-MAA-fibrinogen polyclonal antibodies to MDA-SCHB-modified HSA (containing only MDA adducts). D , quantitative results from the affinity purification of IgG anti-MDA/MAA. Proportion of anti-MDA/MAA among total IgG in healthy controls and RA patients based on amount of purified IgG in different fractions. E , proportion of anti-MAA among IgG anti-MDA/MAA in healthy controls and RA patients. Fib, Fibrinogen; FT, flow through; HSA, human serum albumin; IgG, immunoglobulin G; MAA, malondialdehyde acetaldehyde; MDA, malondialdehyde; RA, rheumatoid arthritis; SCBH, sodium cyanoborohydride.

Article Snippet: To purify MDA/MAA-reactive polyclonal antibodies from human serum, 1 ml Streptavidin Sepharose High Performance resin (Cytiva) was mixed with 1 mg biotinylated and modified full-length fibrinogen for 1.5 h at RT, packed in empty PD-10 columns (Cytiva) and equilibrated with PBS.

Techniques: Purification, Affinity Purification, Modification, Spectrophotometry, Enzyme-linked Immunosorbent Assay