cd33m  (OriGene)

Journal: Scientific reports

Article Title: Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression.

doi: 10.1038/s41598-017-00290-w

Figure Lengend Snippet: Figure 1. Evidence for domain-specific and concentration dependent associations between C1q and CD33 using purified proteins in slot blot assays. Unlabeled proteins were immobilized on membranes; biotin labeled proteins were in solution. (A–C) Dose related binding of immobilized whole C1q and gC1q, but not CLR, to biotin-CD33M (5 ug/ml). (D) Biotin-whole C1q (10 ug/ml) binds to immobilized CD33m (lacking the extracellular V-like domain and expressing the C2-like domain) in a dose specific manner. (HSA = human serum albumin control showing no reactivity. Bound proteins were detected using streptavidin conjugated Infrared 800 (LI-COR). Pixel density was determined by densitometry. (C,D) Bar graphs represent the mean ± SE of pooled data; one-way ANOVA followed by Tukey’s pairwise multiple comparison was used to determine significance. N ≥ 3.

Article Snippet: Recombinant (r) hu proteins purified from cDNA transfected HEK293 T cells were: full length CD33 (CD33M, Novoprotein Scientific Inc., Short Hills, NJ), CD33m (TP317716, Origene technologies, Rockville, MD), biotin-labeled CD33M (CD3-HB22R; ACRO Biosystems, Bethesda, MD).C1q derived from normal hu serum (Complement Technology Inc., Tyler, TX) was used either as unmodified or biotin labeled, or cleaved into gC1q (collagenase digests) or CLR (pepsin digests) as described12, 52.

Techniques: Concentration Assay, Purification, Dot Blot, Labeling, Binding Assay, Expressing, Control, Comparison

Journal: Scientific reports

Article Title: Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression.

doi: 10.1038/s41598-017-00290-w

Figure Lengend Snippet: Figure 2. C1q binds to CD33M and CD33m isoforms on the cell surface. (A) A representative flow cytometry plot showing binding of biotin C1q (20 ug/ml) to HEK293T cells transfected with plasmids encoding CD33m and CD33M and control mock plasmid. Cells were incubated with C1q as outlined in Materials and Methods. Numbers in graphs = % positive/MFI. Debris/dying cells was excluded on the basis of forward and side scatter. (B) Pooled flow cytometry data illustrating binding of C1q (at comparable levels) on the surface of CD33m/M transfected HEK293T cells and a lack of C1q binding on mock transfected cells. For A and B, N = 3. (C) Representative slot blot showing that C1q binds to cell-associated CD33m, CD33M or LAIR-1 in a dose dependent manner. Protein prepared from whole cell lysates (HEK293T cells transfected with plasmid encoding CD33m, CD33M or LAIR-1) was immobilized on membranes at increasing doses; biotin-whole C1q was in solution at 10 ug/ml. HSA = human serum albumin control. Protein concentration was calculated by the BCA method. Bound proteins were detected using streptavidin conjugated Infrared 800 (LI-COR). (D) Pooled data analyses illustrating dose related interactions between biotin-C1q and cell-associated CD33m or CD33M represented in (C). (B,D), the mean ± SE of pooled data is shown; for all proteins and doses, N = 3–5. *P = < 0.05, **P = <0.01, ***P < 0.001. One-way ANOVA followed by Tukey’s pairwise multiple comparison was used.

Article Snippet: Recombinant (r) hu proteins purified from cDNA transfected HEK293 T cells were: full length CD33 (CD33M, Novoprotein Scientific Inc., Short Hills, NJ), CD33m (TP317716, Origene technologies, Rockville, MD), biotin-labeled CD33M (CD3-HB22R; ACRO Biosystems, Bethesda, MD).C1q derived from normal hu serum (Complement Technology Inc., Tyler, TX) was used either as unmodified or biotin labeled, or cleaved into gC1q (collagenase digests) or CLR (pepsin digests) as described12, 52.

Techniques: Flow Cytometry, Binding Assay, Transfection, Control, Plasmid Preparation, Incubation, Dot Blot, Protein Concentration, Comparison

Journal: Scientific reports

Article Title: Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression.

doi: 10.1038/s41598-017-00290-w

Figure Lengend Snippet: Figure 3. C1q triggers CD33 ITIM phosphorylation and CD33-LAIR-1 physical associations in human monocytes. Monocytes were either untreated (utx), treated with whole C1q, gC1q, C1q CLR or pervanadate (PV) as detailed in Materials and Methods. (A,B) Immunoblot analyses showing increased phosphorylated (p) CD33 after treatment with C1q and gC1q, but not C1q CLR. CD33 was immunoprecipitated with anti-CD33M antibody; tyrosine phosphorylation of CD33 (pCD33M) was detected with anti-phosphotyrosine (4G10) antibody. The membrane was stripped and re-probed with anti-CD33 WM53 antibody (CD33M). Arrows denote molecular weight of 67kD. Bar graphs denote pooled data expressed as fold change relative to total CD33 and corresponding statistical analyses. One-way ANOVA with post-hoc Tukey multiple comparisons was used to determine significance. N = 4 for A; N = 3 for B. (C) The addition of whole C1q (20 ug/ml) prompts concurrent increases in pLAIR-1 and pCD33 in a phospho-immunoreceptor array. (D) C1q CLR “tails” (20 ug/ml) do not elicit increases in pCD33M in the phospho-immunoreceptor array. Bar graphs represent fold change (plus/minus C1q), calculated from the mean pixel density of duplicates, as determined by densitometry analysis. Corresponding C1q minus/plus array data is located below each bar graph in C and D. Data from a typical array are shown. N = 3 for C and D. (E) Proximity ligation assay, performed on freshly isolated human monocytes as detailed in materials and methods, showing that whole C1q is required for CD33-LAIR-1 crosslinking. Red fluorescent dots represent molecular associations between CD33 and LAIR-1; blue represents nuclear staining with DAPI. CLR and gC1q represent the C1q collagen tail and globular heads of C1q, respectively. Original magnification = 60X. One of three representative experiments is shown.

Article Snippet: Recombinant (r) hu proteins purified from cDNA transfected HEK293 T cells were: full length CD33 (CD33M, Novoprotein Scientific Inc., Short Hills, NJ), CD33m (TP317716, Origene technologies, Rockville, MD), biotin-labeled CD33M (CD3-HB22R; ACRO Biosystems, Bethesda, MD).C1q derived from normal hu serum (Complement Technology Inc., Tyler, TX) was used either as unmodified or biotin labeled, or cleaved into gC1q (collagenase digests) or CLR (pepsin digests) as described12, 52.

Techniques: Phospho-proteomics, Western Blot, Immunoprecipitation, Membrane, Molecular Weight, Proximity Ligation Assay, Isolation, Staining

Journal: Scientific Reports

Article Title: Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression

doi: 10.1038/s41598-017-00290-w

Figure Lengend Snippet: Evidence for domain-specific and concentration dependent associations between C1q and CD33 using purified proteins in slot blot assays. Unlabeled proteins were immobilized on membranes; biotin labeled proteins were in solution. ( A–C ) Dose related binding of immobilized whole C1q and gC1q, but not CLR, to biotin-CD33M (5 ug/ml). ( D ) Biotin-whole C1q (10 ug/ml) binds to immobilized CD33m (lacking the extracellular V-like domain and expressing the C2-like domain) in a dose specific manner. (HSA = human serum albumin control showing no reactivity. Bound proteins were detected using streptavidin conjugated Infrared 800 (LI-COR). Pixel density was determined by densitometry. (C , D) Bar graphs represent the mean ± SE of pooled data; one-way ANOVA followed by Tukey’s pairwise multiple comparison was used to determine significance. N ≥ 3.

Article Snippet: Anti-human (hu) antibodies (Abs) used in flow cytometry, proximity ligation assays (PLA) and immunoprecipitation assays were commercially obtained and included: Anti-LAIR-1 (mAb DX26; BD Pharmingen; rabbit polyclonal, Sigma HPA011155); mAb anti-CD33m (HIM3-4; BD-Pharmingen, Biolegend); mAb anti-CD33M WM53; BD Biosciences, Biolegend); mAb anti-CD14 (BD Pharmingen); rabbit polyclonal anti-CD14 (Abgent Inc.) anti-RAGE (rabbit polyclonal; BIOSS, mAb MM0520-8D11; Abcam); anti-p33 (mAb74.5.2: Santa Cruz Biotech).

Techniques: Concentration Assay, Purification, Dot Blot, Labeling, Binding Assay, Expressing

Journal: Scientific Reports

Article Title: Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression

doi: 10.1038/s41598-017-00290-w

Figure Lengend Snippet: C1q binds to CD33M and CD33m isoforms on the cell surface. ( A ) A representative flow cytometry plot showing binding of biotin C1q (20 ug/ml) to HEK293T cells transfected with plasmids encoding CD33m and CD33M and control mock plasmid. Cells were incubated with C1q as outlined in Materials and Methods. Numbers in graphs = % positive/MFI. Debris/dying cells was excluded on the basis of forward and side scatter. ( B ) Pooled flow cytometry data illustrating binding of C1q (at comparable levels) on the surface of CD33m/M transfected HEK293T cells and a lack of C1q binding on mock transfected cells. For A and B, N = 3. ( C ) Representative slot blot showing that C1q binds to cell-associated CD33m, CD33M or LAIR-1 in a dose dependent manner. Protein prepared from whole cell lysates (HEK293T cells transfected with plasmid encoding CD33m, CD33M or LAIR-1) was immobilized on membranes at increasing doses; biotin-whole C1q was in solution at 10 ug/ml. HSA = human serum albumin control. Protein concentration was calculated by the BCA method. Bound proteins were detected using streptavidin conjugated Infrared 800 (LI-COR). ( D ) Pooled data analyses illustrating dose related interactions between biotin-C1q and cell-associated CD33m or CD33M represented in ( C) . ( B , D ), the mean ± SE of pooled data is shown; for all proteins and doses, N = 3–5. *P = < 0.05, **P = <0.01, ***P < 0.001. One-way ANOVA followed by Tukey’s pairwise multiple comparison was used.

Article Snippet: Anti-human (hu) antibodies (Abs) used in flow cytometry, proximity ligation assays (PLA) and immunoprecipitation assays were commercially obtained and included: Anti-LAIR-1 (mAb DX26; BD Pharmingen; rabbit polyclonal, Sigma HPA011155); mAb anti-CD33m (HIM3-4; BD-Pharmingen, Biolegend); mAb anti-CD33M WM53; BD Biosciences, Biolegend); mAb anti-CD14 (BD Pharmingen); rabbit polyclonal anti-CD14 (Abgent Inc.) anti-RAGE (rabbit polyclonal; BIOSS, mAb MM0520-8D11; Abcam); anti-p33 (mAb74.5.2: Santa Cruz Biotech).

Techniques: Flow Cytometry, Binding Assay, Transfection, Plasmid Preparation, Incubation, Dot Blot, Protein Concentration

Journal: Scientific Reports

Article Title: Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression

doi: 10.1038/s41598-017-00290-w

Figure Lengend Snippet: C1q triggers CD33 ITIM phosphorylation and CD33-LAIR-1 physical associations in human monocytes. Monocytes were either untreated (utx), treated with whole C1q, gC1q, C1q CLR or pervanadate (PV) as detailed in Materials and Methods. ( A , B ) Immunoblot analyses showing increased phosphorylated (p) CD33 after treatment with C1q and gC1q, but not C1q CLR. CD33 was immunoprecipitated with anti-CD33M antibody; tyrosine phosphorylation of CD33 (pCD33M) was detected with anti-phosphotyrosine (4G10) antibody. The membrane was stripped and re-probed with anti-CD33 WM53 antibody (CD33M). Arrows denote molecular weight of 67kD. Bar graphs denote pooled data expressed as fold change relative to total CD33 and corresponding statistical analyses. One-way ANOVA with post-hoc Tukey multiple comparisons was used to determine significance. N = 4 for A; N = 3 for B. ( C ) The addition of whole C1q (20 ug/ml) prompts concurrent increases in pLAIR-1 and pCD33 in a phospho-immunoreceptor array. ( D ) C1q CLR “tails” (20 ug/ml) do not elicit increases in pCD33M in the phospho-immunoreceptor array. Bar graphs represent fold change (plus/minus C1q), calculated from the mean pixel density of duplicates, as determined by densitometry analysis. Corresponding C1q minus/plus array data is located below each bar graph in C and D. Data from a typical array are shown. N = 3 for C and D. ( E ) Proximity ligation assay, performed on freshly isolated human monocytes as detailed in materials and methods, showing that whole C1q is required for CD33-LAIR-1 crosslinking. Red fluorescent dots represent molecular associations between CD33 and LAIR-1; blue represents nuclear staining with DAPI. CLR and gC1q represent the C1q collagen tail and globular heads of C1q, respectively. Original magnification = 60X. One of three representative experiments is shown.

Article Snippet: Anti-human (hu) antibodies (Abs) used in flow cytometry, proximity ligation assays (PLA) and immunoprecipitation assays were commercially obtained and included: Anti-LAIR-1 (mAb DX26; BD Pharmingen; rabbit polyclonal, Sigma HPA011155); mAb anti-CD33m (HIM3-4; BD-Pharmingen, Biolegend); mAb anti-CD33M WM53; BD Biosciences, Biolegend); mAb anti-CD14 (BD Pharmingen); rabbit polyclonal anti-CD14 (Abgent Inc.) anti-RAGE (rabbit polyclonal; BIOSS, mAb MM0520-8D11; Abcam); anti-p33 (mAb74.5.2: Santa Cruz Biotech).

Techniques: Western Blot, Immunoprecipitation, Molecular Weight, Proximity Ligation Assay, Isolation, Staining

Journal: Scientific Reports

Article Title: Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression

doi: 10.1038/s41598-017-00290-w

Figure Lengend Snippet: Masking of CD33C2 domains impedes C1q binding on the cell surface. ( A , B ) Increased detection of the CD33C2, but not the CD33V domain or LAIR-1, occurs after sialidase treatment of THP-1 cells. Cells were treated with sialidase (20 mU/10 6 cells) for 60 min at 37 °C in a humidified incubator, resuspended twice in PBS, then in staining buffer for detection of CD33C2 domains (HIM3-4), CD33V domains (WM53) and LAIR-1. A = Pooled data, N ≥ 4. B = Representative experiment from A; gates were set according to isotype Ab controls. ( C ) Unmasking of CD33C2 domains with sialidase correlates with increased binding of C1q on THP-1 cells; N = 3–4. ( D ) Reduced levels of sialic acid on the THP-1 surface after sialidase treatment as revealed by decreased binding to Sambucus nigra lectin (SNL) (5 ug/ml). N = 5. ( E ) Pooled flow cytometry data showing highly expressed CD33C2 and CD33V domains and LAIR-1 on freshly isolated normal monocytes. Results represent mean +/− SEM. N = 3–4. For ( A , C , D ) solid bars = no sialidase; open bars = sialidase. Two-tailed Student’s t test was used to determine significance.

Article Snippet: Anti-human (hu) antibodies (Abs) used in flow cytometry, proximity ligation assays (PLA) and immunoprecipitation assays were commercially obtained and included: Anti-LAIR-1 (mAb DX26; BD Pharmingen; rabbit polyclonal, Sigma HPA011155); mAb anti-CD33m (HIM3-4; BD-Pharmingen, Biolegend); mAb anti-CD33M WM53; BD Biosciences, Biolegend); mAb anti-CD14 (BD Pharmingen); rabbit polyclonal anti-CD14 (Abgent Inc.) anti-RAGE (rabbit polyclonal; BIOSS, mAb MM0520-8D11; Abcam); anti-p33 (mAb74.5.2: Santa Cruz Biotech).

Techniques: Binding Assay, Staining, Flow Cytometry, Isolation, Two Tailed Test

Journal: Scientific Reports

Article Title: Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression

doi: 10.1038/s41598-017-00290-w

Figure Lengend Snippet: Maturing DCs exhibit decreases in LAIR-1 and CD14 along with a reduced ability to detect CD33C2 epitopes. ( A , B ) Decreased expression of LAIR-1 and CD14 on DCs. ( C , D ) Pooled flow cytometry data showing CD33C2 domains (detected with mAb HIM3-4) and CD33V domains (detected with mAb WM53) on freshly isolated monocytes and DCs obtained after 5–6 days of culture with 50 U/ml rGM-CSF and 50 ng/ml rIL-4. ( E ) Pooled data showing co-detection of CD33C2 and CD33V domains on fresh monocytes and day 6 DCs. ( F ) Representative dot plot comparing co-detection of CD33C2/V epitopes on freshly isolated monocytes and day 6 DCs; gates were set based on isotype controls. (G ) Increased levels of sialic acid on immature DCs detected with Sambucus nigra lectin (SNL) linked to PECy7. For ( A – E ), N = 3–5; for (G), N = 2. Two-tailed Student’s t test was used to determine significance. Solid bars represent day 0; hatched bars represent day 6.

Article Snippet: Anti-human (hu) antibodies (Abs) used in flow cytometry, proximity ligation assays (PLA) and immunoprecipitation assays were commercially obtained and included: Anti-LAIR-1 (mAb DX26; BD Pharmingen; rabbit polyclonal, Sigma HPA011155); mAb anti-CD33m (HIM3-4; BD-Pharmingen, Biolegend); mAb anti-CD33M WM53; BD Biosciences, Biolegend); mAb anti-CD14 (BD Pharmingen); rabbit polyclonal anti-CD14 (Abgent Inc.) anti-RAGE (rabbit polyclonal; BIOSS, mAb MM0520-8D11; Abcam); anti-p33 (mAb74.5.2: Santa Cruz Biotech).

Techniques: Expressing, Flow Cytometry, Isolation, Two Tailed Test

Journal: Scientific Reports

Article Title: Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression

doi: 10.1038/s41598-017-00290-w

Figure Lengend Snippet: Evidence for domain-specific and concentration dependent associations between C1q and CD33 using purified proteins in slot blot assays. Unlabeled proteins were immobilized on membranes; biotin labeled proteins were in solution. ( A–C ) Dose related binding of immobilized whole C1q and gC1q, but not CLR, to biotin-CD33M (5 ug/ml). ( D ) Biotin-whole C1q (10 ug/ml) binds to immobilized CD33m (lacking the extracellular V-like domain and expressing the C2-like domain) in a dose specific manner. (HSA = human serum albumin control showing no reactivity. Bound proteins were detected using streptavidin conjugated Infrared 800 (LI-COR). Pixel density was determined by densitometry. (C , D) Bar graphs represent the mean ± SE of pooled data; one-way ANOVA followed by Tukey’s pairwise multiple comparison was used to determine significance. N ≥ 3.

Article Snippet: Recombinant (r) hu proteins purified from cDNA transfected HEK293 T cells were: full length CD33 (CD33M, Novoprotein Scientific Inc., Short Hills, NJ), CD33m (TP317716, Origene technologies, Rockville, MD), biotin-labeled CD33M (CD3-HB22R; ACRO Biosystems, Bethesda, MD).C1q derived from normal hu serum (Complement Technology Inc., Tyler, TX) was used either as unmodified or biotin labeled, or cleaved into gC1q (collagenase digests) or CLR (pepsin digests) as described , .

Techniques: Concentration Assay, Purification, Dot Blot, Labeling, Binding Assay, Expressing

Journal: Scientific Reports

Article Title: Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression

doi: 10.1038/s41598-017-00290-w

Figure Lengend Snippet: C1q binds to CD33M and CD33m isoforms on the cell surface. ( A ) A representative flow cytometry plot showing binding of biotin C1q (20 ug/ml) to HEK293T cells transfected with plasmids encoding CD33m and CD33M and control mock plasmid. Cells were incubated with C1q as outlined in Materials and Methods. Numbers in graphs = % positive/MFI. Debris/dying cells was excluded on the basis of forward and side scatter. ( B ) Pooled flow cytometry data illustrating binding of C1q (at comparable levels) on the surface of CD33m/M transfected HEK293T cells and a lack of C1q binding on mock transfected cells. For A and B, N = 3. ( C ) Representative slot blot showing that C1q binds to cell-associated CD33m, CD33M or LAIR-1 in a dose dependent manner. Protein prepared from whole cell lysates (HEK293T cells transfected with plasmid encoding CD33m, CD33M or LAIR-1) was immobilized on membranes at increasing doses; biotin-whole C1q was in solution at 10 ug/ml. HSA = human serum albumin control. Protein concentration was calculated by the BCA method. Bound proteins were detected using streptavidin conjugated Infrared 800 (LI-COR). ( D ) Pooled data analyses illustrating dose related interactions between biotin-C1q and cell-associated CD33m or CD33M represented in ( C) . ( B , D ), the mean ± SE of pooled data is shown; for all proteins and doses, N = 3–5. *P = < 0.05, **P = <0.01, ***P < 0.001. One-way ANOVA followed by Tukey’s pairwise multiple comparison was used.

Article Snippet: Recombinant (r) hu proteins purified from cDNA transfected HEK293 T cells were: full length CD33 (CD33M, Novoprotein Scientific Inc., Short Hills, NJ), CD33m (TP317716, Origene technologies, Rockville, MD), biotin-labeled CD33M (CD3-HB22R; ACRO Biosystems, Bethesda, MD).C1q derived from normal hu serum (Complement Technology Inc., Tyler, TX) was used either as unmodified or biotin labeled, or cleaved into gC1q (collagenase digests) or CLR (pepsin digests) as described , .

Techniques: Flow Cytometry, Binding Assay, Transfection, Plasmid Preparation, Incubation, Dot Blot, Protein Concentration

Journal: Scientific Reports

Article Title: Evidence for C1q-mediated crosslinking of CD33/LAIR-1 inhibitory immunoreceptors and biological control of CD33/LAIR-1 expression

doi: 10.1038/s41598-017-00290-w

Figure Lengend Snippet: C1q triggers CD33 ITIM phosphorylation and CD33-LAIR-1 physical associations in human monocytes. Monocytes were either untreated (utx), treated with whole C1q, gC1q, C1q CLR or pervanadate (PV) as detailed in Materials and Methods. ( A , B ) Immunoblot analyses showing increased phosphorylated (p) CD33 after treatment with C1q and gC1q, but not C1q CLR. CD33 was immunoprecipitated with anti-CD33M antibody; tyrosine phosphorylation of CD33 (pCD33M) was detected with anti-phosphotyrosine (4G10) antibody. The membrane was stripped and re-probed with anti-CD33 WM53 antibody (CD33M). Arrows denote molecular weight of 67kD. Bar graphs denote pooled data expressed as fold change relative to total CD33 and corresponding statistical analyses. One-way ANOVA with post-hoc Tukey multiple comparisons was used to determine significance. N = 4 for A; N = 3 for B. ( C ) The addition of whole C1q (20 ug/ml) prompts concurrent increases in pLAIR-1 and pCD33 in a phospho-immunoreceptor array. ( D ) C1q CLR “tails” (20 ug/ml) do not elicit increases in pCD33M in the phospho-immunoreceptor array. Bar graphs represent fold change (plus/minus C1q), calculated from the mean pixel density of duplicates, as determined by densitometry analysis. Corresponding C1q minus/plus array data is located below each bar graph in C and D. Data from a typical array are shown. N = 3 for C and D. ( E ) Proximity ligation assay, performed on freshly isolated human monocytes as detailed in materials and methods, showing that whole C1q is required for CD33-LAIR-1 crosslinking. Red fluorescent dots represent molecular associations between CD33 and LAIR-1; blue represents nuclear staining with DAPI. CLR and gC1q represent the C1q collagen tail and globular heads of C1q, respectively. Original magnification = 60X. One of three representative experiments is shown.

Article Snippet: Recombinant (r) hu proteins purified from cDNA transfected HEK293 T cells were: full length CD33 (CD33M, Novoprotein Scientific Inc., Short Hills, NJ), CD33m (TP317716, Origene technologies, Rockville, MD), biotin-labeled CD33M (CD3-HB22R; ACRO Biosystems, Bethesda, MD).C1q derived from normal hu serum (Complement Technology Inc., Tyler, TX) was used either as unmodified or biotin labeled, or cleaved into gC1q (collagenase digests) or CLR (pepsin digests) as described , .

Techniques: Western Blot, Immunoprecipitation, Molecular Weight, Proximity Ligation Assay, Isolation, Staining