Fig 1: Melanocytes exhibit two high molecular weight complexes containing WASH, only the higher one of which comigrates with strumpellin. (A) Western blot against antistrumpellin, anti-WASH, anti-SWIP, anti-Fam21, anti-CCDC53 and anti-WAFL with anti-a-tubulin loading control of control (CTRL) or strumpellin knockout (Str1 and Str2) immortal melanocyte line lysates. (B) Integrated density analysis of strumpellin, WASH, SWIP, CCDC53 and Fam21 expression in strumpellin knockout immortal melanocyte line lysates (Str1 and Str2) normalized against control (CTRL) immortal melanocyte line lysates (n = 3). (C) Schematic representation of the WASH complex (based on Jia et al., 2010). SWIP is green, Strumpellin (Str) is orange, Fam21 is red, WASH is shown with its VCA domain folded back into the complex in blue and a connecting line between the domains, and CCDC53 is purple. Protein sizes are not to scale. (D) Blue native PAGE Western blot showing antistrumpellin, anti-WASH, anti-SWIP, anti-Fam21, anti-CCDC53 and anti-WAFL in control (CTRL) or strumpellin knockout (Str1) immortal melanocyte line lysates.
Fig 2: WASH- and WAFL-positive vesicles enlarge and cluster near the cell centre in strumpellin knockout melanocytes. (A) Strumpellin f/f Tyr::CreB- (left – CTRL) or Tyr::CreB+ strumpellin knockout (middle and right – Str1 and Str2) immortal melanocyte lines were immunostained against anti-WASH, and inserts are enlargements of the perinuclear region and show enlarged WASH-positive vesicles (arrows). (B) Strumpellin f/f Tyr::CreB- control (left – CTRL) or Tyr::CreB+ strumpellin knockout (middle and right – Str1 and Str2) immortal melanocyte lines were immunostained against anti-WAFL (green) and phalloidin (red), and inserts are enlargements of the perinuclear region and show colocalization between WAFL and actin (arrows). (C) Single-channel WAFL image from B. Arrows show collapsed and enlarged WAFL-positive vesicles present in strumpellin f/f Tyr::CreB+ immortal melanocyte lines. (D) The measure function of ImageJ was used to quantify the fluorescent intensity of whole Control (CTRL) or strumpellin knockout (Str1 and Str2) immortal melanocyte line cells immunostained against anti-WASH (n = 3, a total of 59, 44 and 57 CTRL, Str1 and Str2 cells, respectively, with at least 10 cells per experimental repeat). Graph show means, error bars = SEM, one-way anova was used for statistical analysis: P = <0.0072, Dunn's multiple comparison post-test compared all columns for statistical difference: *P = 0.05, **P = =0.01) (E). Control (CTRL) and strumpellin knockout (Str1 and Str2) immortal melanocyte lines stained with anti-WAFL were scored blind for clustered and enlarged WAFL-positive vesicles (n = 97, 62 and 51 for CTRL, Str1 and Str2 cells, respectively, pooled from three experiments). (F, G). The analysed particle function of ImageJ was used to count the number of WASH-positive vesicles (F) and measure WASH-positive vesicle size (G) in control (CTRL) and strumpellin knockout (Str1 and Str2) immortal melanocyte lines (n = 64, 62 and 42 for CTRL, Str1 and Str2, respectively, pooled from three experiments, graphs show means, error bars = SEM, one-way anova was used for statistical analysis: P = <0.0001 for both F and G, Dunn's multiple comparison post-test compared all columns for statistical difference: *P = 0.05, **P = =0.01, ***P = 0.001). Scale bars are 10 µm.
Fig 3: Strumpellin deletion in the melanocyte linage does not impair hair pigmentation or melanocyte localization to hair follicles in adult mice. (A) Gene targeting strategy to generate strumpellin f/f Tyr::CreB+ mice. Tyr::CreB excises strumpellin in the melanocyte lineage. (B) A genotyping assay for floxed strumpellin in wild-type (left lane), strumpellin f/f (middle lane) and strumpellin wt/f (right lane) mice. (C) Coat colour (top) and hindlimb colour (bottom) in ~3-month-old wild-type (left), strumpellin wt/f (middle) and strumpellin f/f (right) Tyr::CreB+ mice. (D) Dorsal skin of ~8-month-old strumpellin f/f Tyr::CreB- mouse stained by IHC with anti-DCT (melanocytes) in red at ×10 (left) and ×40 (right) magnification. (E) Dorsal skin of ~8-month-old strumpellin f/f Tyr::CreB+ mouse stained by IHC with anti-DCT (melanocytes) in red at ×10 (left) and ×40 (right) magnification. Scale bars for ×10 and ×40 magnification are 200 and 50 µm, respectively.
Supplier Page from Abcam for Anti-Strumpellin antibody