Western blots of DNAJB2 expression in mice and control patients. A: Western blot and expression level of DNAJB2 in skeletal muscle of dystrophic mdx4Cv and control C57BL/6 mice show no significant differences. Expression level was normalized to α-tubulin. B–C: Western blot and relative expression level of DNAJB2, are shown for the different tissues analyzed in mouse (B) and human (C). Positive control is an extract of C2 myoblast cotransfected by DNAJB2 V1 and V2 expression plasmids. D: Western blot and expression level of DNAJB2 in skeletal muscle of patients with sIBM, IBMPFD, and myofibrillar myopathy (myotilin mutation) and of controls. The green arrows indicate the two alternatively spliced isoforms, V1 and V2, of the DNAJB2 protein. DRG, dorsal root ganglion; LV, left ventricle; RV, right ventricle; TA, tibial anterior; Delt, deltoid muscle.

DNAJB2 immunoreactivity in normal control C57BL/6 mice. A–I: Serial transverse (A–C) and longitudinal (D–I) sections of the tibial anterior muscle in wild-type mice. DNAJB2 is expressed at the NMJ, shown by double staining with DNAJB2 (green; A, D, G), α-bungarotoxin (red; B, E, H), and their co-staining (yellow; C, F, I). The branched structure of the NMJ postsynaptic membrane observed in (D–F) is shown at a larger magnification in (G–I). Nuclei are stained with 4,6-diamidino-2-phenylindole (DAPI; blue; C, F, I). Scale bars = 50 μm. J–O: Isolated sciatic nerve is shown on longitudinal (J–L) and transverse (M–O) sections, with anti-neurofilament (red; J, M), DNAJB2 (green; K, N), and double staining (orange-yellow; L, O). The inset represents a larger magnification of a part of the nerve shown in J–L. Bars indicate a magnification of 200 μm (J–L) and 25 μm (M–O). P–U: A small nerve branch on a transverse section through the psoas muscle reveals anti-DNAJB2 immunofluorescence (Q, T) that is weaker than in the NMJ. Axonal colocalization (orange; R, U) of anti-DNAJB2 (green; Q, T) with anti-neurofilament (red; P, S) is found. Scale bars = 50 μm.

Expression of DNAJB2 in dystrophic muscle of the mdx4Cv mice. A: DNAJB2 staining of transverse section of tibial anterior muscle in mdx mouse. The green box in (A) is seen at a higher magnification in (B–C) by H&E (B) and anti-DNAJB2 immunohistochemical stains (C). Similarly, the blue box in (A) is shown at a higher magnification in (D–E) by H&E stain (D) and DNAJB2 immunofluorescence (E). Notice that DNAJB2 is diffusely expressed in the sarcoplasm and at the sarcolemma of small regenerating fibers (blue arrowhead), whereas in slightly larger regenerating fibers its expression is mainly observed at the sarcolemma (red arrowhead). In mdx mouse, strong expression of DNAJB2 is revealed at the neuromuscular junction (green arrowhead in C). DNAJB2 in NMJs is seen further in the purple box (A) and at a higher magnification (F), and by confocal imaging (G–H; DNAJB2 in green. Nuclei are shown in red (propidium iodide)). I–L: Double immunostaining at NMJs is shown: anti-DNAJB2 in green (I), α-bungarotoxin in red (J), α-bungarotoxin merged with DAPI (blue, K) and merged with anti-DNAJB2 (yellow, L). M–P: Co-expression of DNAJB2 and α-bungarotoxin is seen (white arrows) at the sarcolemma of regenerating fibers (DNAJB2, green, M; α-bungarotoxin, red, N; α-bungarotoxin merged with DAPI, blue, O; α-bungarotoxin merged with anti-DNAJB2, yellow (P). Scale bars = 50 μm, except (H) in which the bar corresponds to 25 μm.

DNAJB2 immunoreactivity in Duchenne muscular dystrophy and in protein aggregate myopathies. A–B: The biopsy of a patient with DMD reveals immunoreactivity for anti-DNAJB2 in regenerating fibers (arrows) and in a necrotic fiber (asterisk). Serial transverse sections with H&E (A) and DNAJB2 (B) stains are shown. C–H: Serial sections indicating regenerating fibers (examples indicated with arrows) at stains with antibodies against DNAJB2 (C), without first antibody (D), neonatal myosin (E), vimentin (F), NCAM (G), and utrophin (H). I–J: In a patient with IBMPFD (patient 20) immunoreactivity with anti-DNAJB2 is revealed in the aggregations, in and around the vacuoles, and at the sarcolemma of muscle fibers, as indicated on serial transverse sections with H&E (I) and DNAJB2 (J) stains. K–P: In sIBM patients DNAJB2 immunoreactivity is revealed in some muscle fibers and around vacuoles, as shown on serial sections with H&E (K), DNAJB2 stains (L), and without primary antibody (M) in patient 27, and in aggregations as shown on serial sections with H&E (N), DNAJB2 stains (O) and without primary antibody (P) in patient 25. Q–T: Serial transverse sections in a patient with myofibrillar myopathy caused by a mutation in ZASP (patient 33) reveal immunoreactivity in the aggregates observed with H&E stain (Q) for the antibodies against DNAJB2 (R), αB-crystallin (S), and ubiquitin (T). Scale bars =100 μm.

DNAJB2 immunoreactivity in normal human tissues. A–B: NMJs (arrows) in control muscle (control 9; see Table 1) show strong immunoreactivity for the DNAJB2 antibody, revealed by immunohistochemical (A–B) and immunofluorescence (C) techniques. Co-localization of DNAJB2 with α-bungarotoxin at the neuromuscular junction is shown in yellow (C arrow). D–F: A longitudinal section through a normal sural nerve (control 10) reveals axonal colocalization (yellow) (F) of DNAJB2 (D) and neurofilament (E). One axon is indicated by arrows. G–H: Myotendinous junctions (arrows) in a normal control biopsy (control 2) reveal no immunoreactivity for anti-DNAJB2, as indicated on serial transverse sections at H&E stains (G) and anti-DNAJB2 immunoreactivity (H). I–J: Transverse sections through the ventral horn of the spinal cord (control 12) at H&E (I) and DNAJB2 (J) stains, reveal anti-DNAJB2 immunoreactivity in the soma of motoneurons and in axons. K–L: Normal control skeletal muscle (control 6) shows muscle fibers with a very weak sarcoplasmic immunoreactivity using the anti-DNAJB2 antibody, at serial transverse sections with H&E (K) and DNAJB2 (L) stains. Reactivity for anti-DNAJB2 in the axons (arrow) of a transversally cut perimysial nerve (asterisk) and in the wall of a perimysial blood vessel (§) are shown. M–N: Intrafusal fibers of a muscle spindle are slightly stained with anti-DNAJB2, shown on transverse serial sections through normal control muscle (control 1) by H&E (M) and DNAJB2 (N) stains. O–R: Serial transverse muscle sections (control 6) show a perimysial nerve at H&E stains (O), and fluorescence staining with anti-DNAJB2 (P), anti-neurofilament (Q), and their co-staining (R). The myonuclei are stained in blue (DAPI). Scale bars = 100 μm, except (B, C) in which the bar corresponds to 10 μm.