Fig 1: Pancreatitis preferentially affects lysosome-associated membrane protein (LAMP) C terminus. LAMP levels were measured by immunoblot in (A–C) whole-tissue homogenates and (D) lysosome-enriched fraction from pancreata of mice with cerulein (CR) or choline-deficient, ethionine-supplemented diet (CDE) pancreatitis, using either Abs against the C terminus of LAMP-1 (Abcam) and LAMP-2 (Sigma-Aldrich) or Abs that recognize the luminal part of LAMPs (Iowa Developmental Studies Hybridoma Bank; see Materials and Methods). (C) LAMP band intensity was normalized to that of extracellular signal-activated kinases (ERK) in the same sample, and the mean LAMP/ERK ratio in pancreatitis group was further normalized to that in control (saline) group. Values are mean ± SEM (n = 4 per group). *P < .05 versus control; #P < .05 versus the corresponding LAMP band intensity detected with C-terminal Ab. (D) Red and blue dashed lines indicate a shift in the position of LAMP-2 band.
Fig 2: Pancreatitis does not cause bulk lysosome-associated membrane protein (LAMP) deglycosylation. Cerulein (CR) and l-arginine (Arg) pancreatitis were induced in (A) rats or (B, C) mice. Pancreatic tissue homogenates from control (saline) and pancreatitic animals were treated with PNGase F (F), Jack bean α-mannosidase (J), endoglycosidase H (E), neuraminidase (N), or neuraminidase plus O-glycanase (N/O), as described in Materials and Methods. LAMP-1 and LAMP-2 were analyzed by immunoblot using C-terminal or luminal Abs, as indicated. Numbers to the right are protein molecular mass markers in kDa. (C) Red and blue dashed lines indicate a shift in the position of LAMP-2 band.
Fig 3: Lysosome-associated membrane protein 2 (LAMP-2) deficiency causes macrophage infiltration and stellate cell activation but not fibrosis in pancreas. Immunofluorescent (A), histochemical (B, E), and immunohistochemical (D) staining of pancreatic tissue sections from 6-month-old wild-type and LAMP-2 knockout mice for markers of macrophages (CD68 and CD206), neutrophils [chloroacetate esterase (CAE)], activated stellate cells (a-SMA), and fibrosis (Goldner trichrome and collagen-1). In (A), nuclei stained with 4',6-diamidino-2-phenylindole (DAPI). (C). Electron micrograph showing abundant macrophages (M) in pancreas of a 6-month-old LAMP-2 knockout mouse. (F) Immunoblot for a-SMA (a-smooth muscle actin) in pancreas of wild-type and LAMP-2 knockout mice. (G). a-SMA and collagen-1 were quantified on pancreatic tissue sections as the percentage of the positive area. Values are mean ± SEM (n = 3 per group). *P < .05 versus wild type. Scale bars: 10 µm (A, B, D, E) and 2 µm (C).
Fig 4: Autophagy flux was affected by PA treatment. (A) LC3B-II/I ratio, total LC3B and p62 proteins were increased in PA-dependent manner. (B) Transcription levels of lc3b increased, but p62 was unchanged by PA treatment. The upper panel shows representative electrophorogram of lc3b and p62 bands from RT-PCR and lower panels show the corresponding histograms from real-time qPCR. (C) Co-immunoprecipitation (Co-IP) results indicate that there was decreased association of p62 with LAMP2, p62 with LC3B, and LC3B with LAMP2, but an increased association of p62 with preproinsulin in the PA treatment group. *p<0.05, **p<0.01, ***p<0.001 compared with the control group.
Fig 5: LPS suppresses the formation of autophagosomes without influencing the function of lysosomes. a N9 microglial cells were exposed to 1 μg/mL LPS for 6 h, 12 h, and 24 h. The expressions of SQSTM1, an autophagy substrate, and LAMP2, a lysosome membrane protein, were detected by western blotting and quantified (b, c). d Levels of Cathepsin E (CTSE) were determined by western blotting in microglial cells treated with LPS for 6 h, 12 h, and 24 h, and quantified (e). f N9 microglial cells were treated with 1 μg/mL LPS for 6 h, 12 h, and 24 h, or with 100 nM bafilomycin A1 for 6 h. The fluorescence intensity of LysoSensor was recorded with a 443-nm excitation filter and a 505-nm emission filter. g N9 microglial cells were treated with LPS in the presence or absence of 10 μΜ chloroquine (CQ) for 24 h. The expression of autophagy-related proteins was measured by western blotting and quantified (h, i). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 vs control; #p < 0.05, ##p < 0.01 vs CQ
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