Fig 1: Colocalization of p62 in macrophages during cholestasis.A. Colocalization of p62 and CD68+ macrophages in human PSC. Paraffin embedded formalin fixed tissue sections from normal human PSC liver were analyzed immunohistochemically using a rabbit polyclonal antibody directed against p62, a mouse monoclonal antibody directed against the macrophage marker CD68 followed by FITC 488 conjugated anti-rabbit and Alexa Fluor 594-conjugated anti-mouse secondary antibodies. Figures are representative of hepatic tissue isolated from three Control and four PSC patients respectively, (200X). B-C. Colocalization of p62 and F4/80 positive macrophages in murine cholestasis. Paraffin embedded formalin fixed tissue sections from B. Sham, BDL liver C. WT, Mdr2KO liver were analyzed immunohistochemically using a rabbit polyclonal antibody directed against p62, a rat polyclonal antibody directed against the macrophage marker F4/80 followed by FITC 647 conjugated anti-rabbit and Alexa Fluor 488-conjugated anti-rat secondary antibodies. Slides were examined using fluorescent microscopy (Blue arrows = p62 positive hepatocytes, red arrows = macrophages, yellow arrows-colocalization), nuclei were visualized by DAPI. Figures are representative of hepatic tissue from three each of sham, WT and the BDL, Mdr2KO animals, respectively (200X).
Fig 2: Increased expression of autophagic proteins in PSC.Western blotting of p62, ATG5/12, ATG7, Beclin1, LC3B, LAMP1 and LAMP2 in liver extracts prepared from patients with PSC and Controls. Blots were normalized to GAPDH expression. Data are presented as Mean ± STDEV and were statistically analyzed using Student’s t-test, *p<0.05, **p<0.01, (N = 6/group).
Fig 3: Increased accumulation of p62, LC3b, LAMP1 and LAMP2 in PSC and BDL mice is located primarily in parenchymal cells that surround the portal triad.Tissue sections from normal human liver, PSC, sham and BDL mice were examined for p62, LC3b, LAMP1 and LAMP2 expression. A. Human control and PSC. B. Sham and BDL. N = 3-5/group, 200X, CV-central vein, PT-portal triad. Arrows indicate increased staining. C. 10-week old WT and Mdr2KO (200X). N = 3-5/group, 200X, CV-central vein, PT-portal triad. Arrows indicate increased staining.
Fig 4: Colocalization of p62 and 4-HNE in liver in cholestasis A. Control and PSC liver (200X). B. Sham and BDL cholestatic liver (100X) C. WT and Mdr2KO liver (100X). Arrows indicate periportal hepatocytes of increased staining/colocalization. n = 4/group, Blue-Dapi, Green-4-HNE, Cyan-p62. CV-central vein, PT-portal triad.
Fig 5: p62 is a target of reactive aldehydes in in vitro cell culture and human PSC.Cells were treated with 100μM 4-HNE for 60 min. Cells were lysed, modified proteins were biotinylated using biotin hydrazide and carbonylated proteins purified by streptavidin bead pulldown. Carbonylation of p62 was assessed by Western blotting as described in methods. A. RAW264.7, B. HepG2. C. Carbonylated proteins from control and PSC liver extracts were treated with biotin hydrazide (5mM/60 min) followed by streptavidin purification and p62 Western analysis. N = 3/condition.
Supplier Page from Novus Biologicals, a Bio-Techne Brand for p62/SQSTM1 Antibody [Alexa Fluor® 594]
Available conjugates: Biotin;CoraFluor 1;PerCP;Alexa Fluor 594