Journal: bioRxiv

Article Title: Tissue Kallikrein 1 cleaves complement factor C3 and activates the alternative complement pathway

doi: 10.1101/2025.09.08.674260

Figure Lengend Snippet: Citrated plasma (1 µL) from adult C57BL/6J mice injected with CsA + LPS (pre-injection [pre] and 24 hours post-injection [post]) was resolved on 4-15% SDS-PAGE under reducing conditions and immunoblotted for (A) KLK1 and KLK1+KAL, (B) FB and Ba, and (C) C3 and C3d. (A) Free and KAL-bound KLK1, (B) Ba and FB, and (C) C3d and C3 were quantified. Blue boxes indicate the quantified areas. MW = Molecular weight marker. F = female mouse, M = male mouse.

Article Snippet: These kidney sections then were stained with either biotinylated goat anti-mouse C3d antibody (20 μg/mL; R&D Systems, BAF2655) and streptavidin Alexa Fluor 488 conjugate (5 μg/mL; Invitrogen, S32354), following streptavidin/biotin blocking according to manufacturer’s instructions (Vector Laboratories, SP-2002) to block endogenous biotin and streptavidin binding sites, or goat anti-mouse TM antibody (10 μg/mL; R&D Systems, AF3894) and donkey anti-goat IgG Alexa Flour 488 (5 μg/mL; Invitrogen, A-11055).

Techniques: Clinical Proteomics, Injection, SDS Page, Molecular Weight, Marker

Journal: bioRxiv

Article Title: Tissue Kallikrein 1 cleaves complement factor C3 and activates the alternative complement pathway

doi: 10.1101/2025.09.08.674260

Figure Lengend Snippet: Adult C57BL/6J mice were injected intraperitoneally with CsA (30 mg/kg) + LPS (5 mg/kg). Citrated plasma was collected pre- and 24 hours post-CsA+LPS injection and analyzed by western blotting for KLK1, Ba, and C3d (C3b cleavage product); see Supplemental Figure 1. (A) Free and KAL-bound KLK1 were quantified and the bound fraction relative to the total (free + bound) was calculated. (B) Ba and FB were quantified and the percentage of Ba to total FB (FB + Ba) was calculated. (C) C3d and C3 were quantified and percentage of C3d to total C3 (C3 + C3d) was calculated. Columns = Mean, Error bars = SD, Blue = male, Pink = female. Significance was determined with a paired t-test. * = p <0.05, ** = p <0.01. (D) Ba and (E) C3d negatively correlated with KLK1 (Pearson correlation coefficient, r = −0.6684, p <0.05; Pearson correlation coefficient, r = −0.3914, p >0.2; respectively). (D and E) Gray = pre-CsA + LPS-injected mice, Black = 24 hours post-CsA + LPS-injected mice.

Article Snippet: These kidney sections then were stained with either biotinylated goat anti-mouse C3d antibody (20 μg/mL; R&D Systems, BAF2655) and streptavidin Alexa Fluor 488 conjugate (5 μg/mL; Invitrogen, S32354), following streptavidin/biotin blocking according to manufacturer’s instructions (Vector Laboratories, SP-2002) to block endogenous biotin and streptavidin binding sites, or goat anti-mouse TM antibody (10 μg/mL; R&D Systems, AF3894) and donkey anti-goat IgG Alexa Flour 488 (5 μg/mL; Invitrogen, A-11055).

Techniques: Injection, Clinical Proteomics, Western Blot

Journal: bioRxiv

Article Title: Tissue Kallikrein 1 cleaves complement factor C3 and activates the alternative complement pathway

doi: 10.1101/2025.09.08.674260

Figure Lengend Snippet: Adult C57BL/6J mice were injected intraperitoneally with CsA (30 mg/kg) + LPS (5 mg/kg) or DMSO (control). Citrated plasma and kidneys were collected 24 hours post-injection. Mounted fixed frozen kidney sections were incubated with biotinylated anti-mouse C3d (C3b cleavage product)/streptavidin Alexa Fluor 488 (green, A and C ) or anti-mouse TM/anti-goat IgG Alexa Flour 488 (green, B and D ). Cell nuclei were stained with DAPI (blue). Stained kidney sections were imaged by immunofluorescence microscopy with a 60x objective. Scale bar = 10 micrometers. Plasma sTM was measured by ELISA (E) , and circulating creatinine was measured by enzymatic assay (F) . Columns = Mean, Error bars = SD, Blue = male, Pink = female. Significance was determined with an unpaired t-test. * = p <0.05, ** = p <0.01. (G) sTM positively correlated with creatinine in DMSO- (●) and CsA + LPS-treated (▲) mice. Pearson correlation coefficient, r = 0.8326, p <0.05.

Article Snippet: These kidney sections then were stained with either biotinylated goat anti-mouse C3d antibody (20 μg/mL; R&D Systems, BAF2655) and streptavidin Alexa Fluor 488 conjugate (5 μg/mL; Invitrogen, S32354), following streptavidin/biotin blocking according to manufacturer’s instructions (Vector Laboratories, SP-2002) to block endogenous biotin and streptavidin binding sites, or goat anti-mouse TM antibody (10 μg/mL; R&D Systems, AF3894) and donkey anti-goat IgG Alexa Flour 488 (5 μg/mL; Invitrogen, A-11055).

Techniques: Injection, Control, Clinical Proteomics, Incubation, Staining, Immunofluorescence, Microscopy, Enzyme-linked Immunosorbent Assay, Enzymatic Assay

Journal: Theranostics

Article Title: C5aR1 is a master regulator in Colorectal Tumorigenesis via Immune modulation

doi: 10.7150/thno.45058

Figure Lengend Snippet: The strong correlation between complement activation and CRC. ( A-D ) The associations between the mRNA levels of C3 (A), C5 (B), C5AR1 (C), and C5AR2 (D) and the overall survival of CRC patients (n=364). ( E, F ) The effect of C3 , C5 , C5ar1 or C5ar2 deficiency on AOM/DSS-induced colorectal tumorigenesis compared to WT control. Images of the colorectum, where CRC often developed at distal (rectum) sites (E), and quantitative analysis of tumor number and mass (F). The experiment was duplicated. Scale bar in (E), 1 cm. ( G ) Pathological analysis of control or tumor tissues, including H&E, Ki-67 and C3d staining. Scale bar, 100 µm for H&E and 50 µm for IHC. ( H ) C5a concentration in colon tissue homogenates (CTHs) measured by ELISA. n≥ 7 in each group of WT, C3 -KO, C5 -KO, C5ar1 -KO, or C5ar2 -KO mice. Data are represented as mean ± SEM; ns, not significant; * P <0.05; and *** P <0.001. NC, negative control.

Article Snippet: Then, mouse colon sections were incubated with an affinity purified pAb specific for mouse complement component C3d (1:80; R&D Systems, Minneapolis, MN), rabbit anti-Ki67 antibody (1 μg/ml; Abcam, Cambridge, MA) or rabbit anti-CD8 alpha antibody (1:1500; Abcam, Cambridge, MA) at 4°C overnight.

Techniques: Activation Assay, Control, Staining, Concentration Assay, Enzyme-linked Immunosorbent Assay, Negative Control