Fig 1: a STRING network showing the known interactions between the proteins encoded by the genes differentially expressed in the Manffl/flCol2Cre− and Col2Cre+ cartilage at P5. b Deletion of MANF increases the levels of GRP78 but not GRP94 in cartilage at P5, as shown by Western blotting and densitometry measurement (n = 3). c Western blotting of proteins co-immunoprecipitated in the 293 cells expressing FLAG-tagged recombinant MANF
Fig 2: a BrdU labelling of control primary chondrocytes and chondrocytes treated with tunicamycin with and without exogenous MANF (n = 2). b Western blot and densitometry measurement of GRP78 levels in untreated and tunicamycin-treated primary chondrocytes with and without exogenous MANF (n = 2). Tun tunicamycin. ***P < 0.001, **P < 0.01, *P < 0.05
Fig 3: a Targeting strategy used to generate the Manf−/− (‘knockout first’) and Manffl/fl Col2Cre+ (conditional knockout allele) lines. Black boxes represent Manf exons; LoxP sites are marked by arrowheads and FRT sites by ovals. b Western blotting showing the deletion of MANF in Manf−/− liver at E19.5, GAPDH as loading control. c LacZ staining of the whole mount ß-galactosidase (Rosa26) reporter crossed with Col2Cre line embryo at E15.5, showing cartilage-specific expression of the Col2Cre transgene. d Western blotting showing deletion of MANF in Manffl/fl Col2Cre+ cartilage at P21, GAPDH as loading control. Scale bars 1 mm and 100 μm
Fig 4: a Immunohistochemistry showing the localisation of cartilage extracellular matrix components, type II collagen, type X collagen and matrilin-3 in Manffl/flCol2Cre− and Col2Cre+ growth plates at P21. b 2 h BrdU labelling of proliferation in Manffl/flCol2Cre− and Col2Cre+ growth plates at P21 showing a 29% decrease in chondrocyte proliferation upon deletion of MANF (n = 5). c TUNEL assay per zone in Manffl/flCol2Cre− and Col2Cre+ growth plates at P21 (n = 3). d Total apoptosis in Manffl/flCol2Cre− and Col2Cre+ growth plates at P21 (n = 3). RZ resting zone, PZ proliferative zone, HZ hypertrophic zone, NC negative control. **P < 0.01. Scale bar 200 μm
Fig 5: a X-ray radiographs of Manffl/fl Col2Cre− Matn3+/+, Manffl/fl Col2Cre− Matn3V194D/V194D and Manffl/fl Col2Cre+ Matn3V194D/V194D mice at 6 weeks, showing the dramatic effect of removal of MANF from cartilage of the mouse model of MED. b Western blot and densitometry measurement of intracellular GRP78 levels in Matn3V194D/V194D primary chondrocytes with and without exogenous MANF (n = 4). c Western blot and densitometry measurement of intracellular matrilin-3 levels in Matn3V194D/V194D primary chondrocytes with and without exogenous MANF (n = 4). d Immunocytochemistry for matrilin-3 showing that exogenous MANF has no effect on the intracellular retention of mutant matrilin-3 (in green) in Matn3V194D/V194D primary chondrocytes. DAPI was used as a counterstain. Scale bars 5 mm (a) and 200 μm (d) (colour figure online)
Supplier Page from R&D Systems, a Bio-Techne Brand for Recombinant Human MANF Protein, CF