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Fig. 3. Induction of sRB in non-lymphoid cells. (A) Time-dependent formation of sRB in H[µ ${Delta}$ CH1] induced to synthesize µ ${Delta}$ CH1 chains. After culture in the absence of tetracycline for the indicated periods, cells were harvested, fixed and stained with fluorescent anti-µ; bar 5 µm. The inset in the center panel (day 4) shows a magnification of a cell containing a small µ-positive dot. (B) sRB formation correlates with accumulation of detergent-insoluble µ ${Delta}$ CH1 chains in H[µ ${Delta}$ CH1]. Aliquots of detergent soluble (s) and insoluble (i) material from cells treated as in A were analyzed by western blotting as described in legend to Fig. 1. (C) sRB localize preferentially in the vicinity of the MTOC. H[µ ${Delta}$ CH1] cells cultured for 5 days without tetracycline were simultaneously stained with anti- ${gamma}$ -tubulin and anti-µ antibodies; bar 5 µm. (D) Dose-dependent formation of sRBs imply a threshold concentration for µ ${Delta}$ CH1 condensation. H[µ ${Delta}$ CH1] cells were cultured for 5 days in medium supplemented with decreasing doses of tetracycline as indicated. µ ${Delta}$ CH1 chains were quantified in extracts containing total (TOT, lysis in SDS) or TX100 soluble (SOL) and insoluble (INS) proteins and expressed as arbitrary units. At the tetracycline concentration of 0.25 ng/ml or more, most µ ${Delta}$ CH1 proteins are found in the soluble fraction. At 0.12 ng/ml detergent-insoluble chains become preponderant and continue to increase at lower concentrations, when more µ ${Delta}$ CH1 chains are synthesized.

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