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. 2013 Jul 11;93(1):6–18. doi: 10.1016/j.ajhg.2013.05.004

Figure 3.

Figure 3

KLHL40 Expression in Human and Mouse Tissues

(A) Taqman quantitative real-time PCR analysis of cDNA from adult or fetal human tissues. Error bars represent the SD. The following abbreviation is used: Sk, skeletal.

(B) KLHL40 levels in C2C12 cells and mouse tissues (HPA, top panel) and immunoblotting for sarcomeric α-actin (clone 5C5, middle panel) and GAPDH (lower panel). Lanes are as follows: myo, C212 myoblasts; D2, myotubes on day 2 of differentiation; D4, myotubes on day 4 of differentiation; D6, myotubes on day 6 of differentiation; Gastroc (left), C57BL/6 postnatal day 2 (d2) gastrocnemius; Gastroc (right), C57BL/6 8-week-old gastrocnemius; and EDL (extensor digitorum longus) to liver, C57BL/6 8-week-old tissues. For all mouse tissue lysates, samples were pooled from three different mice.

(C) On the left is KLHL40 expression in human skeletal muscle (HPA, top panel), immunoblotting for α-actinin (clone EA-53, middle panel), and Coomassie staining of MHC band (bottom panel). Lanes are as follows: F:19w, 19-week-old fetus; F:23w, 23-week-old fetus; F:31w, 31-week-old fetus; 11d, 11-day-old neonate; 6mo, 6-month-old baby; and C1–C4, healthy adult controls of 19–42 years of age. On the right, KLHL40 intensity normalized to MHC for fetal muscle is 3.34 ± 0.92 (n = 3) versus 1.37 ± 0.21 (n = 6) for postnatal skeletal muscle. ∗p = 0.023, unpaired two-tailed t test. Error bars represent the SEM.

(D) Single Z-plane confocal microscopy showing localization of KLHL40 (green) and α-actinin (red) in a longitudinal section of skeletal muscle from a 31-week-old fetus; costaining with Hoechst (blue) is also shown (Merge). Scale bars represent 5 μm.

(E) Immunoblotting shows that KLHL40 is absent in KLHL40-associated NEM muscle (II-1 from family 10 [F10] and V-2 from family 17 [F17]) compared with healthy control muscle (C1, C3, and C4). Coomassie staining of the MHC band (bottom panel) and immunoblotting for sarcomeric α-actin (clone 5C5, middle panel) indicate similar or greater loading for the KLHL40-associated NEM samples compared with control samples.

(F) Immunofluorescence for KLHL40 in a human 23-week-old fetal skeletal muscle sample (F:23w), an adult healthy control (C4), and KLHL40-associated NEM muscle biopsies (II-1 from family 10 [F10], V-2 from family 17 [F17], family 21 [F21], and family 26 [F26]). Scale bars represent 50 μm.